In vitro glycosylation of proteins and enzymes

ABSTRACT

The present invention is broadly concerned with new in vitro glycosylation methods that provide rational approaches for producing glycosylated proteins, and the use of glycosylated proteins. In more detail, the present invention comprises methods of glycosylating a starting protein having an amino sidechain with a nucleophilic moiety, comprising the step of reacting the protein with a carbohydrate having an oxazoline moiety on the reducing end thereof, to covalently bond the amino sidechain of the starting protein with the oxazoline moiety, wherein the glycosylated protein substantially retains the structure and function of the starting protein. Target proteins include oxidase, oxidoreductase and dehydrogenase enzymes. The glycosylated proteins advantageously have molecular weights of at least about 7500 Daltons. In a further embodiment, the present invention concerns the use of glycosylated proteins, fabricated by the methods disclosed herein, in the assembly of amperometric biosensors.

RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 16/224,795, filed Dec. 19, 2018, entitled IN VITRO GLYCOSYLATION OFENZYMES AND PROTEIN THERAPEUTICS, which claims the benefit of U.S.Provisional Patent Application Ser. No. 62/607,655, filed Dec. 19, 2017,entitled IN VITRO GLYCOSYLATION OF ENZYMES AND PROTEIN THERAPEUTICS.These applications are incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under ContractHR0011-16-C-0124 awarded by DARPA. The Government has certain rights inthe invention.

BACKGROUND OF INVENTION Field of Invention

The present invention relates to new in vitro glycosylation methods thatprovide rational approaches for generating glycosylated proteins, andthe use thereof.

Description of the Prior Art

Glycosylation is the process by which a carbohydrate is covalentlyattached to a target protein, such as an enzyme.¹⁻² The attachment ofcarbohydrate moieties to proteins is a post-translational modification(PTM) that provides greater proteomic diversity to proteins.Glycosylation is critical for a wide range of biological processes,including cell attachment to the extracellular matrix and protein-ligandinteractions in the cell. In the normal course of in vivo proteinglycosylation, proteins are generally N-glycosylated on asparagine, orO-glycosylated on serine or threonine residues.

The glycosylation of proteins serves various functions.²⁻³ For instance,it is known that some proteins and protein classes do not fold correctlyin the absence of glycosylation. In other instances, proteins are notstable in the absence of oligosaccharides linked at the amide nitrogenof certain asparagine residues).⁴ In addition, it is well establishedthat some enzymes, such as glucose oxidase, display better stability andenzymatic parameters when glycosylated than in the absence ofglycosylation.⁵⁻⁶

The importance of N-linked glycosylation is becoming increasinglyevident in the field of pharmaceuticals.^(2, 4, 7) In recent years, aconsiderable emphasis has been placed on the development and deploymentof in vitro glycosylation methods to alter and enhance the glycoforms onproteins, and in particular, antibodies.⁸ A number of strategies havebeen reported.⁹ One approach that has emerged is a multi-step processthat involves production of an N-linked glycosylated protein, removal ofthe glycan moieties except for the terminal N-acetylglucosamine residuesattached to asparagines, and subsequent enzymatic attachment of a newglycoform to alter the immune response of the target protein. Thismethod of glycan remodeling requires (1) that the carbohydrate to beactivated has an N-acetyl moiety at the 2-position of the reducing endof the carbohydrate to be conjugated, (2) that the target glycosylationsites have a GlcNAc residue already present on the protein, and (3) thecarbohydrate to be conjugated be a substrate forendoglycosidase-catalyzed transglycosylation.⁹⁻¹¹ These requirementslimit the modification of proteins utilizing this method. Recently,carbohydrate oxazolines have been used as activated glycosyl donorsubstrates for endoglycosidase-catalyzed transglycosylation.¹² Inaddition to the previously mentioned requirements, the naturallyoccurring hydrolytic activity of endoglycosidases towards the oxazolineslimits the efficiency of this approach.^(7-9, 11)

The in vivo N-glycosylation of lysine residues, i.e. attachment of aglycan to the terminal nitrogen of a lysine sidechain, in full-lengthproteins is rare.¹³ Reports of N-glycosylation of lysine with glucoseare largely associated with modifications of collagen in aged ordiabetic tissue.¹⁴⁻¹⁵ The in vivo attachment of other glycans to lysineremains undescribed. O-glycosylation of hydroxylysine has been reportedand is also commonly associated with collagen formation.¹⁶⁻¹⁷ In thisinstance, the lysine sidechain is post-translationally modified byhydroxylation, which provides the site for glycan attachment. While invivo glycosylation of arginines and histidines have been reported, nocorresponding selective in vitro glycosylation methodology has beenreported.^(13, 18-20)

Other references of interest include those where smaller molecularweight amino acid chains are glycosylated by the oxazolineapproach.^(11-12, 21-26) Much, if not most, of the existing art teachesaway from direct lysine glycosylation by carbohydrate oxazolines infavor of methods in which the carbohydrate oxazoline is used as asubstrate for an endo-β-N-acetylglucosaminidases to synthesize aspartateN-linked glycoproteins.^(11-12, 21-26) Direct lysine glycosylation bycarbohydrate oxazolines are often referred to as “by-products”.²³

SUMMARY OF THE INVENTION

The present invention is broadly concerned with new in vitroglycosylation methods that provide rational approaches for producingglycosylated proteins, and the use of glycosylated proteins. In moredetail, the present invention comprises methods of glycosylating astarting protein having an amino sidechain with a nucleophilic moiety,comprising the step of reacting the protein with a carbohydrate havingan oxazoline moiety on the reducing end thereof, in a compatible aqueousmedium, to covalently bond the amino sidechain of the starting proteinwith the oxazoline moiety, wherein the glycosylated proteinsubstantially retains the structure and function of the startingprotein.

In another embodiment, the invention is concerned with glycosylatedproteins comprising a starting protein having an amino sidechain with anucleophilic moiety glycosylated with a carbohydrate having an oxazolinemoiety on the reducing end thereof, wherein the oxazoline moiety iscovalently bound with the nucleophilic moiety. The glycosylated proteinsadvantageously have molecular weights of at least about 7500 Daltons,and substantially retain the structure and function of the startingprotein.

In a further embodiment, the present invention concerns the use ofglycosylated proteins, fabricated by the methods disclosed herein, inthe assembly of amperometric biosensors. The amperometric biosensorscomprise a counter electrode, a reference electrode, one or moreoptional rejection layers, and a working electrode that comprises asensing element, which comprises a support having a surface; and a layeron the surface, wherein the layer comprises an amino sidechainglycosylated enzyme, wherein the enzyme is predominantly in its activeform.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representative SDS-PAGE gel (8% gel, 1 ug of sample perlane) demonstrating the conjugation of malate dehydrogenase (MDH, lanes1, 3 and 5) with the oxazolines of N-acetylglucosamine (GlcNAc, lane 2),N-acetylmannosamine (ManNAc, lane 4), and N-acetlygalactosamine (GalNAc,lane 6), wherein the protein to carbohydrate ratio in the conjugationreaction was about 1:1000 and the conjugation reaction was allowed toproceed for about 24 hours at room temperature;

FIG. 2 is a representative SDS-PAGE gel (8% gel, 2 ug of sample perlane) demonstrating the conjugation of malate dehydrogenase (MDH, lane2) and L-lactate dehydrogenase (LDH, lane 5) with the oxazolines oftriacetylchitotriose (TACT, lanes 3 and 6, respectively) and hyaluronicacid of nominal molecular weight of about 50 kD (HA50, lanes 4 and 7,respectively), wherein the protein to carbohydrate ratio in theconjugation reaction was about 1:100 and the conjugation reaction wasallowed to proceed for about 24 hours at room temperature;

FIG. 3 is a representative SDS-PAGE gel (8% gel, 2 ug of sample perlane) demonstrating the conjugation of L-lactate oxidase (LOx, lane 2)and sarcosine oxidase (SOx, lane 5) with the oxazolines oftriacetylchitotriose (TACT, lanes 3 and 6, respectively) and hyaluronicacid of nominal molecular weight of about 50 kD (HA50, lanes 4 and 7,respectively), wherein the protein to carbohydrate ratio in theconjugation reaction was about 1:100 and the conjugation reaction wasallowed to proceed for about 24 hours at room temperature;

FIG. 4 is a representative SDS-PAGE gel (8% gel, 2 ug of sample perlane) demonstrating the conjugation of cortisol dehydrogenase (CDH, lane2) and alcohol dehydrogenase (ADH, lane 5) with the oxazolines oftriacetylchitotriose (TACT, lanes 3 and 6, respectively) and hyaluronicacid of nominal molecular weight of about 50 kD (HA50, lanes 4 and 7,respectively), wherein the protein to carbohydrate ratio in theconjugation reaction was about 1:100 and the conjugation reaction wasallowed to proceed for about 24 hours at room temperature;

FIG. 5 is a representative SDS-PAGE gel (8% gel, 2 ug of sample perlane) demonstrating the conjugation of alcohol oxidase (AOx, lane 2)with the oxazolines of triacetylchitotriose (TACT, lane 3) andhyaluronic acid of nominal molecular weight of about 50 kD (HA50, lane4), wherein the protein to carbohydrate ratio in the conjugationreaction was about 1:100 and the conjugation reaction was allowed toproceed for about 24 hours at room temperature;

FIG. 6 is a representative SDS-PAGE gel (12% gel, 0.5 ug of sample perlane) demonstrating the conjugation of malate dehydrogenase (MDH,lane 1) and sarcosine oxidase (SOx, lane 3) with the oxazolines of LewisB tetrasaccharide (LewB4, lanes 2 and 4, respectively), wherein theprotein to carbohydrate ratio in the conjugation reaction was about1:1000 and the conjugation reaction was allowed to proceed for about 72hours at room temperature;

FIG. 7 is a representative SDS-PAGE gel (8% gel, 1 ug of sample perlane) demonstrating the conjugation of malate dehydrogenase (MDH, lanes3, 6 and 9) with the oxazolines of N-acetylglucosamine (GlcNAc, lane 4),N-propanylglucosamine (lane 5), N-n-butanylglucosamine (lane 7),N-isopropanylglucosamine (lane 8), N-2,2,dimethylpropanylglucosamine(lane 10), N-((2-oxoethoxy)-propanyl)glucosamine (lane 11), wherein theprotein to carbohydrate ratio in the conjugation reaction was about1:1000 and the conjugation reaction was allowed to proceed for about 24hours at room temperature;

FIG. 8 is a representative SDS-PAGE gel (8% gel, 0.5 ug of sample perlane) demonstrating the conjugation of sarcosine oxidase (SOx, lanes 2and 4) with the oxazolines of native N-glycan core Man-3 (lane 3) andwith high mannose N-glycan core Man-5 (lane 5), respectively, andwherein the protein to carbohydrate ratio in the conjugation reactionwas about 1:1000 and the conjugation reaction was allowed to proceed forabout 24 hours at room temperature;

FIG. 9 is a representative SDS-PAGE gel (8% gel, 1 ug of sample perlane) demonstrating the conjugation of sarcosine oxidase (SOx, lanes 2,4 and 6) with the oxazolines of triacetylchitotriose (TACT, lanes 3, 5,and 7) at pH 6, 7 and 8, respectively, wherein the protein tocarbohydrate ratio in the conjugation reaction was about 1:1000 and theconjugation reaction was allowed to proceed for about 24 hours at roomtemperature;

FIG. 10 is a representative triplicate mass specific activitydetermination of wild-type native glucose oxidase (GOx, column 1), GOxconjugated with GlcNAc (column 2), GOx conjugated with TACT (column 3),GOx conjugated with HA6 (column 4), and GOx conjugated with HA50 (column5);

FIG. 11 is a representative triplicate mass specific activitydetermination of wild-type native L-lactate oxidase (LOx, column 1), LOxconjugated with GlcNAc (column 2), LOx conjugated with TACT (column 3),LOx conjugated with HA6 (column 4), and LOx conjugated with HA50 (column5);

FIG. 12 is a representative triplicate mass specific activitydetermination of wild-type native NctB oxidase (NicOx, column 1), NicOxconjugated with GlcNAc (column 2), NicOx conjugated with TACT (column3), NicOx conjugated with HA6 (column 4), and NicOx conjugated with HA50(column 5);

FIG. 13 is a representative triplicate mass specific activitydetermination of wild-type native alcohol oxidase (AOx, column 1), AOxconjugated with GlcNAc (column 2), AOx conjugated with TACT (column 3),AOx conjugated with HA6 (column 4), and AOx conjugated with HA50 (column5);

FIG. 14 is a representative triplicate mass specific activitydetermination of wild-type native cortisol dehydrogenase (CortDH, column1), CortDH conjugated with GlcNAc (column 2), CortDH conjugated withTACT (column 3), CortDH conjugated with HA6 (column 4), and CortDHconjugated with HA50 (column 5);

FIG. 15 is a representative triplicate mass specific activitydetermination of wild-type native L-lactate dehydrogenase (LDH, column1), LDH conjugated with GlcNAc (column 2), LDH conjugated with TACT(column 3), LDH conjugated with HA6 (column 4), and LDH conjugated withHA50 (column 5);

FIG. 16 is a representative triplicate mass specific activitydetermination of wild-type native alcohol dehydrogenase (ADH, column 1),ADH conjugated with GlcNAc (column 2), ADH conjugated with TACT (column3), ADH conjugated with HA6 (column 4), and ADH conjugated with HA50(column 5);

FIG. 17 is a representative mass specific activity determination ofwild-type native glucose oxidase (GOx, column 1), GOx conjugated withGlcNAc (column 2), GOx conjugated with TACT (column 3), GOx conjugatedwith HA6 (column 4), and GOx conjugated with HA50 (column 5) after 96hours of incubation at 42 C;

FIG. 18 is a representative fluorescence thermal shift assay (FTSA)comparison of wild-type native glucose oxidase (GOx, circles, Tm=72.0C), GOx conjugated with GlcNAc (squares, Tm=72.4 C), GOx conjugated withTACT (up-triangles, Tm=72.1 C), and GOx conjugated with HA6(down-triangles, Tm=71.8 C);

FIG. 19 is a representative fluorescence thermal shift assay (FTSA)comparison of wild-type native L-lactate oxidase (LOx, circles, Tm=72.0C), LOx conjugated with GlcNAc (squares, Tm=72.4 C), LOx conjugated withTACT (up-triangles, Tm=72.1 C), LOx conjugated with HA6 (down-triangles,Tm=71.8 C), and LOx conjugated with HA50 (diamonds, Tm=72.7 C);

FIG. 20 is a representative fluorescence thermal shift assay (FTSA)comparison of wild-type native alcohol dehydrogenase (ADH, triangles,Tm=61.4 C), ADH conjugated with HA6 (triangles, Tm=58.5 C), and ADHconjugated with HA50 (triangles, Tm=62.5 C);

FIG. 21 is a schematic drawing of the working electrode of aprototypical amperometric biosensor.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides methods of modifying a protein, themethods comprise in vitro glycosylation methods that provide a rationalapproach for generating glycosylated versions of proteins. Thisinvention allows for the custom control of the carbohydrate moietiesplaced on the protein including those that may be stabilizing. Incertain embodiments, the protein is an enzyme. In general, glycosylatedenzymes may be more stable than their unglycosylated counterparts.

In one aspect of this invention, the target protein is unglycosylated.The new glycosylation method does not require the use of eithereukaryotic expression platforms or the presence of existing N-linkedglycosylation sites in the target protein. In a unique aspect of thisinvention, the target protein may already be N-glycosylated orO-glycosylated in the traditional sense, thereby providing ahyper-glycosylated protein product. This method of in vitro glycoproteinmanufacturing may provide a more direct, robust, and scalable methodthan those currently employed, such as enzymatic modification ofexisting glycans or genetic manipulation of the cellular glycosylationmachinery. Existing approaches may suffer from poor scalability and ahigh degree of difficulty. These problems are absent in the presentinvention.

This invention describes a new method for glycosylating proteins thatdoes not require the presence of a sequeon or glycosyltransfer consensusglycosylation amino acid recognition sequence.^(1, 3, 13) In oneembodiment, this invention uses the NE-amino group of the lysinesidechain as a nucleophile to covalently bond to a carbohydrateoxazoline to form one or more amidine moieties (Schemes 1 and 2). Themethod is mild, and allows for the transfer of multiple glycans to atarget protein. A protein may contain one or more lysine residues thatare available to undergo modification by glycosylation.

Scheme 1 shows the numbering of the parent N-acyl-2-amino carbohydratedepicting the free reducing end at C1 (i.e. a hemiacetylic hydroxyl).The dehydration of the N-acyl-2-amino moiety and the C1 hydroxyl formsthe carbohydrate oxazoline in aqueous media. Dehydration can beaccomplished in basic media using published methods.^(12, 21-22) Basessuch as triethylamine and solid Na₃PO₄ may be used to promotedehydration. All carbohydrate manipulations may take place in aqueoussolution, including buffered aqueous solutions, in the absence of anyprotecting group chemistries. In one embodiment, this first reaction offorming a carbohydrate oxazoline takes place over about 24 hours. In apreferred embodiment, this first reaction of forming a carbohydrateoxazoline takes place from about one to six hours.

In one embodiment, a non-obvious neutralization step of the oxazolinemodified glycan with bicine (or any suitable acid or buffer within apreferred pH range) prior to coupling to the target protein (e.g. anenzyme), is required. Most proteins, and in particular enzymes, are notgenerally tolerant of pH values much higher than pH of about 8.5, atwhich point structural unfolding, and covalent modifications begin tooccur. Since formation of the oxazoline requires significant quantities(7.5 molar excess) of base, said base must be either removed byfiltration and/or neutralized with a concentrated buffer solution toprevent exposing the oxazoline or the target protein to a pH above 8.5when the two species are combined. In a preferred embodiment, the pH ofthe oxazoline containing solution is adjusted to maintain optimal enzymecatalytic function. The preferred pH range for the reaction of theoxazoline with a protein target, such as an enzyme, is pH 5 to pH 9. Amore preferred pH range is pH 6.5 to pH 8.5. The most preferred range ispH 7.0 to pH 8.0. Reactions are driven to completion by the action of LeChatliere's Principle regardless of the pH.

Prototypical reaction of the parent N-acyl-2-amino carbohydrateoxazoline with a nucleophilic amino sidechain moiety on a protein yieldsthe carbohydrate modified protein through the two tautomeric bondingschemes as depicted in Scheme 2. These tautomers may be in equilibriumwith each other and other structures related to these two canonicalstructures.¹¹ In one embodiment, the amino acids that contain anucleophilic amino sidechain moiety are selected from the groupconsisting of lysine, arginine and histidine. In a preferred embodiment,the amino acid that contains a nucleophilic amino sidechain moiety islysine. In one embodiment, this second reaction takes place from about 2hours to about 96 hours. In another embodiment, the temperature of thissecond reaction is from about 0° C. to about 37° C. In a preferredembodiment, the temperature of this second reaction is from about 4° C.to about 25° C.

In another embodiment, the invention includes the steps of firstreacting a carbohydrate having an N-acyl-2-amino group on the reducingend thereof with a dehydrating agent to form an oxazoline moiety on thereducing end of a carbohydrate, and then reacting said oxazolinecarbohydrate with a starting protein. In a preferred embodiment, themethod of claim 13, including the step of carrying out said firstreaction for a period from about 1-6 hours, and carrying out saidprotein/oxazoline carbohydrate reaction for a period of from about 2hours to 96 hours.

There is no stereochemical requirement at C2, C3, C4, C5 and C6 of thecarbohydrate, and the N-acyl moiety may be either equatorially oraxially disposed and still result in formation of an oxazoline. The Rmoiety on the acyl group is defined herein.

Thus, in one embodiment of this invention, carbohydrates with anN-acylglucosamine saccharide at the reducing end can covalently bondwith a target protein (Scheme 2). In another embodiment of thisinvention, carbohydrates with an N-acylmannosamine saccharide at thereducing end can covalently bond with a target protein (Scheme 2). Inanother embodiment of this invention, carbohydrates with anN-acylgalactosamine saccharide at the reducing end can covalently bondwith a target protein (Scheme 2). In another embodiment of theinvention, the R group is not limited to methyl (Scheme 2).

One of ordinary skill in the art will recognize that the C3, C4 and C6hydroxyl moieties on the parent N-acyl-2-amino carbohydrate may befurther substituted with other carbohydrates to form disaccharidestructures, and that free C2, C3, C4 and C6 hydroxyls withindisaccharide structures may be further substituted to form largeroligosaccharides. The scope of R₁, R₂ and R₃ is defined herein. R₁, R₂and R₃ are independently selected from the group consisting of H andsaccharides.

The carbohydrate or saccharide according to the present invention is notlimited by its source or origin, and encompasses those obtained fromnatural origins including those from human and other mammalian sources,those produced by genetically engineered animal cells, plant cells,microorganisms, and other cells, those enzymatically manufactured, thosemanufactured by fermentation processes, those artificially synthesizedby chemical processes and others. The carbohydrate or saccharide mayencompass monosaccharides, disaccharides, oligosaccharides, andpolysaccharides, so long as the reducing end saccharide moiety isunprotected on C1 (i.e. contains a hemiacetalic hydroxyl) and containsan N-acyl-2-amino moiety. Examples of N-acyl-2-amino monosaccharidesinclude, but are not limited to, N-acetylglucosamine, N-acylglucosamine,N-acetylgalactosamine, N-acylgalactosamine, N-acetylmannosamine,N-acylmannosamine, N-acetyllallosamine, N-acylallosamine,(2S,3S,4R,5R,6R)-5-acetamido-3,4,6-trihydroxytetrahydro-2H-pyran-2-carboxylicacid,(2S,3R,4R,5R,6R)-5-acetamido-3,4,6-trihydroxytetrahydro-2H-pyran-2-carboxylicacid,(2S,3S,4S,5R,6R)-5-acetamido-3,4,6-trihydroxytetrahydro-2H-pyran-2-carboxylicacid,(2R,3S,4R,5R,6R)-5-acetamido-3,4,6-trihydroxytetrahydro-2H-pyran-2-carboxylicacid,(2S,3S,4R,5R,6R)-5-acyl-3,4,6-trihydroxytetrahydro-2H-pyran-2-carboxylicacid,(2S,3R,4R,5R,6R)-5-acyl-3,4,6-trihydroxytetrahydro-2H-pyran-2-carboxylicacid,(2S,3S,4S,5R,6R)-5-acyl-3,4,6-trihydroxytetrahydro-2H-pyran-2-carboxylicacid,(2R,3S,4R,5R,6R)-5-acyl-3,4,6-trihydroxytetrahydro-2H-pyran-2-carboxylicacid,(2S,3S,4R,5R,6R)-5-(2-(carboxymethoxy)acetamido)-3,4,6-trihydroxytetrahydro-2H-pyran-2-carboxylicacid,2-(2-oxo-2-(((2R,3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)ethoxy)aceticacid,(2S,3R,4R,5R,6R)-5-(2-(carboxymethoxy)acetamido)-3,4,6-trihydroxytetrahydro-2H-pyran-2-carboxylicacid,2-(2-oxo-2-(((2R,3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)ethoxy)aceticacid,(2S,3S,4S,5R,6R)-5-(2-(carboxymethoxy)acetamido)-3,4,6-trihydroxytetrahydro-2H-pyran-2-carboxylicacid,2-(2-oxo-2-(((2R,3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)ethoxy)aceticacid,(2R,3S,4R,5R,6R)-5-(2-(carboxymethoxy)acetamido)-3,4,6-trihydroxytetrahydro-2H-pyran-2-carboxylicacid,2-(2-oxo-2-(((2R,3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)ethoxy)aceticacid, and derivatives and modified derivatives thereof. In a preferredembodiment, the monosaccharide has a molecular weight of at least about200 Daltons to about 500 Daltons.

Examples of disaccharides include, but are not limited to,N-acetylactosamine, N-acylactosamine, N,N′-diacetyl chitobiose,N,N′-diacyl chitobiose, hyaluronic acid disaccharide, keratindisaccharide, keratin sulfate disaccharide, chondroitin disaccharide,chondroitin sulfate disaccharide, dermatan disaccharide, dermatansulfate disaccharide, heparin disaccharide, and derivatives thereof solong as the reducing end of the disaccharide is unprotected on C1 andcontains an N-acyl-2-amino moiety. The disaccharides of the presentinvention may further include those disaccharides that are isolated orsynthesized wherein any C3, C4 or C6 free hydroxyl of an N-acyl-2-aminomonosaccharide is further substituted with a single monosaccharide (atR₁, R₂ or R₃ in Schemes 1 and 2). Such monosaccharides include, but arenot limited to, N-acyl 2-amino monosaccharides, glucose, galactose,allose, mannose, idose, fructose, fucose, xylose, arabinose, glucuronicacid, iduronic acid, neuraminic acid, sialic acid, and derivatives andmodified derivatives thereof.

The oligosaccharides of the current invention encompass those moleculescomposed of three or more monosaccharide units linked together at anyfree hydroxyl in the ordinary sense, either in a linear or branchedmanner, and are usually composed of 3 to 30 monosaccharide units, but insome instances may contain thousands of monosaccharide units. Inparticular, the oligosaccharides of the present invention may furtherinclude those oligosaccharides that are isolated or synthesized whereinany C3, C4 or C6 free hydroxyl of an N-acyl-2-amino monosaccharide issubstituted with a single monosaccharide (depicted as R₁, R₂ and R₃ inFIGS. 1 and 2 ), and any other C2, C3, C4 or C6 free hydroxyl may befurther substituted with one or more monosaccharides. Examples of theoligosaccharide include those discovered in a wide range of organismssuch as animals, plants (including seaweeds), insects, microorganisms,and others, and include, but are not limited to, homooligomers ofN-acetylglucosamine, chitin, partially deacetylated chitin, and otherN-acyl-2-amino monosaccharides, heterooligomers composed of two or moredifferent monosaccharides independently selected from the groupsconsisting of glucose, galactose, mannose, glucosamine, fructose,N-acyl-2-amino monosaccharides, iduronic acid, neuraminic acid, andsialic acid so long as the reducing end unit is unprotected (i.e.contains a hemiacetalic hydroxyl) and contains an N-acyl-2-amino moiety.Other representative oligosaccharides include hyaluronic acid, keratin,keratin sulfate, chondroitin, chondroitin sulfate, dermatan, dermatansulfate, heparin, N-acetyllactosamine oligosaccharide,N-acetylchitotriose, N-acetylchitotetraose, and N-acetylchitopentose,chitin, including partially deacetylated chitin, native N-glycan cores,high-mannose N-glycans, hybrid N-glycans, complex N-glycans, andderivatives thereof.

Examples of native N-glycan cores include, but are not limited to, Man3and Man3F. Examples of high-mannose N-glycans include, but are notlimited to, Man-5, Man-6, Man-7, Man-8, and Man-9. Examples of hydridN-glycans include, but are not limited, those carbohydrates wherein theGlcNAc terminates one branch of the N-glycan core, and a mannoseterminates another branch of the N-glycan core. Examples of complexN-glycans include, but are not limited to, GO-N, GO, GOF-N, GOF, GOFB,G1, G1F, G2, G2F, G2FB, G1S1(α2,3), G1S1(α2,6), G1FS1(α2,3),G1FS1(α2,6), G2S1(α2,3), G2S1(α2,6), G2FS1(α2,3), G2FS1(α 2,6), G2S2(α2,3), G2S2(α2,6), G2FS2(α2,3), G2FS2(α2,6), G2F with 2-α-Gal, A3, G3,G3S3(α2,6), A4, G4, Lewis A, Lewis B, Lewis X, Lewis Y, sialyl Lewis A,and sialyl Lewis X. The nomenclature used to describe the N-glycanswould be known to one skilled in the art. With the exception of theLewis antigens, all native N-glycan cores, high-mannose N-glycans,hybrid N-glycans, and complex N-glycans contain a Man₃GlcNAc₂ core,which may be optionally fucosylated or xylosylated.

The term “modified carbohydrate” (or “modified derivative thereof”) usedherein may refer to those modified through any process of isolation,separation and purification from naturally-occurring sources andorigins, those that have been enzymatically modified, those that havebeen chemically modified, those that have been modified by biochemicalmeans, including microorganisms, wherein such modifications may comprisethose known in the field of glycoscience, for example, hydrolysis,oxidation, reduction, esterification, acylation, amination,etherification, nitration, dehydration, glycosylation, phosphorylation,and sulfation.

The applicable C1-hemiacetal hydroxyl and N-acyl-2-amino-bearingcarbohydrate used as the starting material in implementing the presentinvention include carbohydrate molecules having an N-acylamino group atposition 2 on the reducing end side. Preferred carbohydrates have anacetamido group at position 2 on the reducing end side of amonosaccharide, including, for example, N-acetylglucosamine,N-acetylgalactosamine, and N-acetylmannosamine, and oligosaccharides inwhich the reducing end is selected from N-acetylglucosamine,N-acetylgalactosamine, and other N-acyl-2-amino monosaccharides. In caseof disaccharides and oligosaccharides, preferred examples of thecarbohydrate include N-acetylactosamine, N,N′-diacetylchitobiose,hyaluronic acid disaccharide, other glycosaminoglycan disaccharides.Preferred oligosaccharides include hyaluronic acid, keratin, keratinsulfate, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate,heparin, poly-N-acetyllactosamine, N-acetylchitotriose,N-acetylchitotetraose, and N-acetylchitopentose, chitin, includingpartially deacetylated chitin, high-mannose N-glycans, hybrid N-glycans,and complex N-glycans. In a more preferred embodiment, the linearoligosaccharide is hyaluronic acid.

Linear oligosaccharides such as chitin, including partially deacetylatedchitin, hyaluronic acid, keratin, keratin sulfate, chondroitin,chondroitin sulfate, dermatan, dermatan sulfate, and heparin can achievemolecular weights as much as 5,000,000 Daltons. Preferred sizes of theseoligosaccharides include, but are not limited to, those with molecularweights of at least about 500 Daltons, at least about 1,000 Daltons, atleast about 1,500 Daltons, at least about 3,500 Daltons, at least about5,000 Daltons, at least about 10,000 Daltons, at least about 20,000Daltons, at least about 25,000 Daltons, at least about 33,000 Daltons,at least about 50,000 Daltons, and at least about 100,000 Daltons. Inanother embodiment, preferred sizes of these oligosaccharides include,but are not limited to, those molecular weights that are less than about6,000 Daltons, and less than about 50,000 Daltons.

Branched oligosaccharides such as high-mannose N-glycans, hybridN-glycans, and complex N-glycans have molecular weights that aretypically under 10,000 Daltons. Preferred sizes of theseoligosaccharides include, but are not limited to, those with molecularweights of from about 500 Daltons to about 10,000 Daltons. A morepreferred size of these oligosaccharides is from about 800 Daltons toabout 5,000 Daltons. A most preferred size of these oligosaccharides isfrom about 800 Daltons to about 4500 Daltons.

Each R is individually and independently C1-C6 alkyl, branched C3-C8alkyl, —(CH₂)_(m)—CN, —(CH₂)_(m)OR6, —(CH₂)_(m)—CO₂H, —(CH₂)_(m)—CO₂R6,—(CH₂)_(m)—NR6(R7), —(CH₂)_(m)—S(O)_(n)—C1-C6 alkyl,—(CH₂)_(m)—C(O)NR6(R7), —(CH₂)_(m)—CO₂—C4-C6 heterocyclyl,—(CH₂)_(m)—C4-C6 heterocyclyl, —(CH₂)_(m)—CO₂—C4-C6 heteroaryl, or—(CH₂)_(m)—C4-C6-heteroaryl, wherein each alkyl may optionally containan ether linkage and, wherein each alkyl is optionally substituted withone or two C1-C6 alkyl;

each R6 and R7 is individually and independently H, C1-C6 alkyl, orbranched C3-C8 alkyl;

each m is individually and independently 1, 2, 3, 4, or 5;

each n is individually and independently 0, 1, or 2;

Structural, chemical and stereochemical definitions are broadly takenfrom IUPAC recommendations, and more specifically from Glossary of Termsused in Physical Organic Chemistry (IUPAC Recommendations 1994) assummarized by Müller, P. Pure Appl. Chem. 1994, 66, pp. 1077-1184 andBasic Terminology of Stereochemistry (IUPAC Recommendations 1996) assummarized by Moss, G. P. Pure Appl. Chem. 1996, 68, pp. 2193-2222.

For convenience, certain terms employed in the specification, examplesand claims are collected here. Unless defined otherwise, all technicaland Scientific terms used in this dis closure have the same meanings ascommonly understood by one of ordinary skill in the art to which thisdisclosure belongs. The initial definition provided for a group or termprovided in this disclosure applies to that group or term throughout thepresent disclosure in

The term “alkyl” as used herein refers to a straight chain alkyl,wherein alkyl chain length is indicated by a range of numbers. Inexemplary embodiments, “alkyl” refers to an alkyl chain as defined abovecontaining 1, 2, 3, 4, 5, or 6 carbons (i.e., C1-C6 alkyl). Examples ofan alkyl group include, but are not limited to, methyl, ethyl, propyl,butyl, pentyl, and hexyl.

The term “branched alkyl” as used herein refers to an alkyl chainwherein a branching point in the chain exists, and the total number ofcarbons in the chain is indicated by a range of numbers. In exemplaryembodiments, “branched alkyl” refers to an alkyl chain as defined abovecontaining 3, 4, 5, 6, 7, or 8 carbons (i.e., branched C3-C8 alkyl).Examples of a branched alkyl group include, but are not limited to,iso-propyl, iso-butyl, secondary-butyl, and tertiary-butyl.

The term “heterocycle” or “heterocyclyl” as used herein refers to acyclic hydrocarbon, wherein at least one of the ring atoms is an O, N,or S, wherein the number of ring atoms is indicated by a range ofnumbers. Heterocyclyl moieties as defined herein have C or N bondingsites. For example, in some embodiments, a ring N atom from theheterocyclyl is the bonding atom of the heterocylic moiety. In exemplaryembodiments, “heterocyclyl” refers to a cyclic hydrocarbon as describedabove containing 4, 5, or 6 ring atoms (i.e., C4-C6 heterocyclyl).Examples of a heterocycle group include, but are not limited to,aziridine, oxirane, thirane, azetidine, oxetane, thietane, pyrrolidine,tetrahydrofuran, pyran, thiopyran, thiomorpholine, thiomorpholineS-oxide, thiomorpholine S-dioxide, oxazoline, tetrahydrothiophene,piperidine, tetrahydropyran, thiane, imidazolidine, oxazolidine,thiazolidine, dioxolane, dithiolane, piperazine, oxazine, dithiane, anddioxane.

The term “heteroaryl” as used herein refers to a cyclic hydrocarbon,where at least one of the ring atoms is an O. N. or S, the ring ischaracterized by delocalized at electrons (aromaticity) shared among thering members, and wherein the number of ring atoms is indicated by arange of numbers. Heteroaryl moieties as defined herein have C or Nbonding hands. For example, in some embodiments, a ring N atom from theheteroaryl is the bonding atom of the heteroaryl moiety. In exemplaryembodiments, “heteroaryl” refers to a cyclic hydrocarbon as describedabove containing 5 or 6 ring atoms (i.e., C5-C6 heteroaryl). Examples ofa heteroaryl group include, but are not limited to, pyrrole, furan,thiene, oxazole, thiazole, isoxazole, isothiazole, imidazole, pyrazole,oxadiazole, thiadiazole, triazole, tetrazole, pyridine, pyrimidine,pyrazine, pyridazine, and triazine. The term “substituted in connectionwith a moiety as used herein refers to a further substituent which isattached to the moiety at any acceptable location on the moiety. Unlessotherwise indicated, moieties can bond through a carbon, nitrogen,oxygen, sulfur, or any other acceptable atom.

The term “substituted” in connection with a moiety as used herein refersto a further substituent which is attached to the moiety at anyacceptable location on the moiety. Unless otherwise indicated, moietiescan bond through a carbon, nitro gen, oxygen, sulfur, or any otheracceptable atom.

The term “tautomer” as used herein refers to compounds produced by thephenomenon where in a proton of one atom of a molecule shifts to anotheratom. See March, Advanced Organic Chemistry: Reactions, Mechanisms andStructures, 4th Ed., John Wiley & Sons, pp. 69-74 (1992). Tautomerism isdefined as isomerism of the general form:

G-X—Y═Z→X═Y—Z-G

where the isomers (called tautomers) are readily interconvertible; theatoms connecting the groups X, Y and Z are typically any of C, H, O, orS, and G is a group which becomes an electrofuge or nucleofuge duringisomerization. The most common case, when the electrofuge is H⁺, is alsoknown as “prototropy”. Tautomers are defined as isomers that arise fromtautomerism, independent of whether the isomers are isolable.

The present invention is suitable for the modification of proteins, andmay find application in a variety of situations. More specifically, thisinvention is suitable for the modification of enzymes. Preferred enzymeclasses are oxidase proteins, oxidoreductase proteins, dehydrogenaseproteins, and combinations thereof.

Within the classification of oxidase proteins, the preferred targets arethose used in human health monitoring applications. Oxidase enzymes thatmay benefit from glycosylation using this invention include, but are notlimited to, malate oxidases, EC 1.1.3.3, hexose oxidases, EC 1.1.3.5,aryl-alcohol oxidases, EC 1.1.3.7, L-gulonolactone oxidases, EC 1.1.3.8,pyranose oxidases, EC 1.1.3.10, L-sorbose oxidases, EC 1.1.3.11,pyridoxine 4-oxidases, EC 1.1.3.12, (S)-2-hydroxy-acid oxidases, EC1.1.3.15, ecdysone oxidases, EC 1.1.3.16, secondary-alcohol oxidases, EC1.1.3.18, 4-hydroxymandelate oxidases, EC 1.1.3.19, long-chain-alcoholoxidases, EC 1.1.3.20, thiamine oxidases, EC 1.1.3.23, hydroxyphytanateoxidases, EC 1.1.3.27., N-acylhexosamine oxidases, EC 1.1.3.29,polyvinyl-alcohol oxidases, EC 1.1.3.30, D-Arabinono-1,4-lactoneoxidases, EC 1.1.3.37, vanillyl-alcohol oxidases, EC 1.1.3.38,D-mannitol oxidases, EC 1.1.3.40, alditol oxidases, EC 1.1.3.41, cholinedehydrogenase, EC 1.1.99.1, gluconate 2-dehydrogenase EC 1.1.99.3,glucooligosaccharide oxidases, EC 1.1.99.B3, alcohol dehydrogenase, EC1.1.99.8, cellobiose dehydrogenase, EC 1.1.99.18, aldehyde oxidases, EC1.2.3.1, glyoxylate oxidases, EC 1.2.3.5, indole-3-acetaldehydeoxidases, aryl-aldehyde oxidases, EC 1.2.3.9, retinal oxidases, EC1.2.3.11, abscisic-aldehyde oxidases, EC 1.2.3.14, aldehyde ferredoxinoxidoreductase, EC 1.2.7.5, indolepyruvate ferredoxin oxidoreductase, EC1.2.7.8, aldehyde dehydrogenase, EC 1.2.99.7, dihydroorotate oxidases,EC 1.3.3.1, acyl-CoA oxidases, EC 1.3.3.6, dihydrouracil oxidases, EC1.3.3.7, tetrahydroberberine oxidases, EC 1.3.3.8, tryptophanalpha,beta-oxidases, EC 1.3.3.10, L-galactonolactone oxidases, EC1.3.3.12, acyl-CoA dehydrogenase, EC 1.3.99.3,Isoquinoline-1-oxidoreductase, EC 1.3.99.16, quinaldate4-oxidoreductase, EC 1.3.99.18, D-aspartate oxidases, EC 1.4.3.1,L-amino-acid oxidases, EC 1.4.3.2, monoamine oxidases, EC 1.4.3.4,pyridoxal 5′-phosphate synthase, EC 1.4.3.5, D-glutamate oxidases, EC1.4.3.7, ethanolamine oxidases, EC 1.4.3.8; putrescine oxidases, EC1.4.3.10, cyclohexylamine oxidases, EC 1.4.3.12, protein-lysine6-oxidases, EC 1.4.3.13, D-glutamate (D-aspartate) oxidases, EC1.4.3.15, L-lysine 6-oxidases, EC 1.4.3.20, primary-amine oxidases, EC1.4.3.21, 7-chloro-L-tryptophan oxidases, EC 1.4.3.23,N-methyl-L-amino-acid oxidases, EC 1.5.3.2, non-specific polyamineoxidases, EC 1.5.3.B2, N8-acetylspermidine oxidases(propane-1,3-diamine-forming), EC 1.5.3.B3, N6-methyl-lysine oxidases,EC 1.5.3.4, polyamine oxidases (propane-1,3-diamine-forming), EC1.5.3.B4, N1-acetylpolyamine oxidases, EC 1.5.3.B5, spermine oxidases,EC 1.5.3.B6, pipecolate oxidases, EC 1.5.3.7, dimethylglycine oxidases,EC 1.5.3.10, polyamine oxidases, EC 1.5.3.11, Dihydrobenzophenanthridineoxidases, EC 1.5.3.12, NAD(P)H oxidases, EC 1.6.3.1, urate oxidases, EC1.7.3.3; 3-aci-nitropropanoate oxidases, sulfite oxidases, EC 1.8.3.1,methanethiol oxidases, EC 1.8.3.4; prenylcysteine oxidases, EC 1.8.3.5,L-ascorbate oxidases, EC 1.10.3.3, 3-hydroxyanthranilate oxidases, EC1.10.3.5, rifamycin-B oxidases, EC 1.10.3.6, superoxide dismutase, EC1.15.1.1, reticuline oxidases, EC 1.21.3.3, lactate oxidases, L-EC1.1.3.15, D-amino acid oxidases, EC 1.4.3.3, (S)-6-hydroxynicotineoxidases, EC 1.5.3.5, (R)-6-hydroxynicotine oxidases, EC 1.5.3.6,alcohol oxidases, EC 1.1.3.13, pyruvate oxidases, EC 1.2.3.3, glucoseoxidases, EC 1.1.3.4), L-glutamate oxidases, EC 1.4.3.11, acyl coenzymeA oxidases, EC 1.3.3.6, choline oxidases, EC 1.1.3.17, glutathionesulfhydryl oxidases, EC 1.8.3.3, glycerolphosphate oxidases, EC1.1.3.21, sarcosine oxidases, EC 1.5.3.1, xanthine oxidases, EC1.1.3.22, oxalate oxidases, EC 1.2.3.4, co-factor(s)=Mn²⁺; cholesteroloxidases, EC 1.1.3.6, gamma-glutamyl-putrescine oxidases, EC undefined,obtained from Escherichia coli K12, capable of oxidizing GABA; GABAoxidases, EC undefined, obtained from: Penicillium sp. KAIT-M-117,histamine oxidases (diamine oxidases), EC 1.4.3.22, nucleoside oxidases,EC 1.1.3.39, L-lysine oxidases, EC 1.4.3.14, L-aspartate oxidases, EC1.4.3.16, glycine oxidases, EC 1.4.3.19, galactose oxidases, EC 1.1.3.9,laccases (EC 1.1.3.4), tyrosinases (1.14.18.1), sulfite oxidases,tyramine oxidases, and NADH oxidases (1.11.1.1).

Preferred members of the oxidase class of enzymes include, but are notlimited to, lactate oxidases (EC 1.1.3.15), D-amino acid oxidases (EC1.4.3.3), (S)-6-hydroxynicotine oxidases (EC 1.5.3.5), xanthine (EC1.5.3.6), alcohol oxidases (EC 1.1.3.13), pyruvate oxidases (EC1.2.3.3), glucose oxidases (EC 1.1.3.4), glutamate oxidases (EC1.4.3.11), acyl coenzyme A oxidases (EC 1.3.3.6), choline oxidases (EC1.1.3.17), glutathione sulfhydryl oxidases (EC 1.8.3.3),glycerolphosphate oxidases (EC 1.1.3.21), sarcosine oxidases (EC1.5.3.1), xanthine oxidases (EC 1.1.3.22), oxalate oxidases (EC1.2.3.4), cholesterol oxidases (EC 1.1.3.6), gamma alpha-butyric acid(GABA) oxidases (EC undefined), histamine oxidases (diamine oxidases, EC1.4.3.22), nucleoside oxidases (EC 1.1.3.39), L-lysine oxidases (EC1.4.3.14), L-aspartate oxidases (EC 1.4.3.16), glycine oxidases (EC1.4.3.19), NADH oxidases (EC 1.11.1.1) and galactose oxidases (EC1.1.3.9).

Most preferred members of the oxidase class of enzymes include, but arenot limited to, lactate oxidases (EC 1.1.3.15), alcohol oxidases (EC1.1.3.13), glucose oxidases (EC 1.1.3.4), glutamate oxidases (EC1.4.3.11), choline oxidases (EC 1.1.3.17), sarcosine oxidases (EC1.5.3.1), xanthine oxidases (EC 1.1.3.22), oxalate oxidases (EC1.2.3.4), cholesterol oxidases (EC 1.1.3.6), histamine oxidases (diamineoxidases, EC 1.4.3.22), glycine oxidases (EC 1.4.3.19), NADH oxidases(EC 1.11.1.1) and galactose oxidases (EC 1.1.3.9).

Dehydrogenase enzymes may benefit from glycosylation using thisinvention. Within the classification of dehydrogenase proteins, thepreferred targets are those used human health monitoring applicationsinclude, but are not limited to, alcohol dehydrogenases (EC 1.1.1.1),glucose hydrogenases (EC 1.1.1.47, L-lactate dehydrogenases (1.1.1.27),D-lactate dehydrogenases (1.1.1.28), cortisol dehydrogenases(1.1.1.146), glutamate dehydrogenases (1.4.1.2), fructose dehydrogenases(EC 1.1.99.11), glycerol 3-phosphate dehydrogenases (1.1.1.8),formaldehyde dehydrogenases (EC 1.1.1.284 and EC 1.2.1.46), aldehydedehydrogenases (EC 1.2.1.5), alanine dehydrogenases (EC 1.4.1.1),formate dehydrogenases (EC 1.2.1.2), galactose hydrogenases (EC1.1.1.48), glycerol dehydrogenases (EC 1.1.1.6), glucose-6-phosphatedehydrogenases (EC 1.1.1.49), 3-hydroxybutyrate dehydrogenases (EC1.1.1.30), 3-alpha-hydroxysteroide dehydrogenases (EC 1.1.1.50),isocitrate dehydrogenases (EC 1.1.1.42), inositol dehydrogenases (EC1.1.18), L-leucine dehydrogenases (EC 1.4.1.9), L-malate dehydrogenases(EC 1.1.1.37), and sorbitol dehydrogenases (EC 1.1.1.14).

Preferred members of the dehydrogenase class of enzymes include, but arenot limited to, alcohol dehydrogenases (EC 1.1.1.1), glucosehydrogenases (EC 1.1.1.47, L-lactate dehydrogenases (1.1.1.27), cortisoldehydrogenases (1.1.1.146), glutamate dehydrogenases (1.4.1.2),galactose hydrogenases (EC 1.1.1.48), glycerol dehydrogenases (EC1.1.1.6), glucose-6-phosphate dehydrogenases (EC 1.1.1.49),3-hydroxybutyrate dehydrogenases (EC 1.1.1.30), L-malate dehydrogenases(EC 1.1.1.37), and sorbitol dehydrogenases (EC 1.1.1.14).

Most preferred members of the dehydrogenase class of enzymes include,but are not limited to, alcohol dehydrogenases (EC 1.1.1.1), glucosehydrogenases (EC 1.1.1.47, L-lactate dehydrogenases (1.1.1.27), cortisoldehydrogenases (1.1.1.146), galactose hydrogenases (EC 1.1.1.48),glycerol dehydrogenases (EC 1.1.1.6), glucose-6-phosphate dehydrogenases(EC 1.1.1.49), 3-hydroxybutyrate dehydrogenases (EC 1.1.1.30), L-malatedehydrogenases (EC 1.1.1.37), and sorbitol dehydrogenases (EC 1.1.1.14).

The present invention is also suitable for use with enzymes that aremodified with fusion partners to aid in expression, stabilization,and/or solubility. Fusion partners to the oxidase, oxidoreductase anddehydrogenase proteins include, but are not limited to, greenfluorescent protein (GFP), glutathione S-transferase (GST),maltose-binding protein (MBP), iGg binding domain of Protein G (GB1),Protein A (PA), thioredcoxin (TRX), cyclomaltodextrin glucanotransferase(CMDGT), small ubiquitin-like modifier (SUMO), galactose binding protein(GBP), cellulose binding domain (CBD), calmodulin binding peptide (CBP),bacterial transcription termination/anti termination protein (NusA),growth differentiating factor-8 (GDF8), and ubiquitin (Ub). One skilledin the art will recognize that said fusion proteins may be N-terminal orC-terminal to the catalytic domains of the oxidase, oxidoreductase anddehydrogenase proteins, and may include an optional linker between thetwo protein domains.

In another embodiment, the attachment of a N-acyl-2-amino carbohydrateoxazoline with a nucleophilic amino sidechain moiety on a protein yieldsthe carbohydrate modified protein with an alternative glycosylationpattern to existing, natural glycosylation patterns. This alteredglycosylation pattern has the potential of conferring new properties tothe target protein, such as enhanced stability of an enzyme. Inaddition, the ability to add oligosaccharides to proteins that arenaturally glycosylated, such as glucose oxidase, offers the possibilityto hyper-glycosylate said proteins. This is especially important for thestabilization of enzymes, most especially oxidase, oxidoreductase, anddehydrogenase enzymes, that may be useful in human health monitoringapplications. Another embodiment of this invention allows for themodification a protein without the removal of native glycans, saidnative glycans that are known to be stabilizing to a protein.

Existing N-linked glycosylation involves the use of asparagine sidechains as the attachment point for the glycan moiety. In one embodimentof this invention, sidechains that are traditionally non-glycosylatedsuch as lysine, arginine, and histidine are glycosylated. Since thisstrategy is orthogonal to natural glycosylation methods, proteins, suchas glucose oxidase that are natively glycosylated, can be furtherglycosylated to produce a hyper-glycosylated entity that may havebeneficial characteristics including stabilization and controlledimmunogenicity. Many important proteins are naturally glycosylated, andcannot be further glycosylated on other sites within the properly foldedprotein structure using natural and prior art methods. This inventionprovides a rational method to further glycosylate said proteins, therebystabilizing glycoproteins, such as glucose oxidase, without compromisingthe structure and function, i.e. catalytic ability, of the protein.

One skilled in the art will recognize that protein structure is thethree-dimensional arrangement of atoms in one or more amino acid-chainmolecules. To be able to perform biological functions, proteins foldinto one or more specific spatial conformations driven by a number ofnon-covalent interactions including hydrogen bonding, ionicinteractions, Van der Waals forces, and hydrophobic packing, therebyforming an ensemble. A protein may undergo reversible structural changesin performing its function, which for an enzyme includes catalyticaction on a substrate or substrates to produce a product or products. Aprotein will not function without the correct structure. For example, anenzyme is in its substantially active form when it is able to catalyze aphysical or physiochemical process, and the results of this process areobservable. An enzyme is unable to perform catalysis when the structureis altered such that the enzyme is no longer able to facilitate one ormore steps of the catalytic process, and is therefore deemed inactive.Activity and function depend on the proper specific spatialconformations being occupied, and observed activity is a directindication of proper structure. Thus, modifications of a protein,including enzymes, that do not substantially alter function bydefinition do not substantially alter specific spatial conformations.Changes to function without changes to protein structure are mutuallyexclusive. The stability and therefore activity of a glycosylated orhyper-glycosylated protein relative to the starting protein can beimproved, as reflected by improved longevity of the modified protein'sactivity. This can be quantified by kinetic and biophysical studies,including accelerated enzymatic stability studies at elevatedtemperatures herein. In one embodiment, the stabilized enzyme retains atleast about 25% more activity compared to the starting enzyme inaccelerated stability studies. In a preferred embodiment, the stabilizedenzyme retains at least about 33% more activity compared to the startingenzyme in accelerated stability studies. In a more preferred embodiment,the stabilized enzyme retains at least about 50% more activity comparedto the starting enzyme in accelerated stability studies. In a mostpreferred embodiment, the stabilized enzyme retains at least about 75%more activity compared to the starting enzyme in accelerated stabilitystudies.

In another embodiment, the present invention is a glycosylated proteincomprising a starting protein having an amino sidechain with anucleophilic moiety glycosylated with a carbohydrate having an oxazolinemoiety on the reducing end thereof, said oxazoline moiety covalentlybound with said nucleophilic moiety, said glycosylated protein having amolecular weight of at least about 7500 Daltons, said glycosylatedprotein substantially retaining the structure and function of saidstarting protein. More specifically, the present glycosylated protein isa glycosylated enzyme. Preferred enzyme classes are oxidase proteins,oxidoreductase proteins, dehydrogenase proteins, and combinationsthereof defined herein.

In a further embodiment, the glycosylated protein utilizes the aminosidechain of the starting protein that is selected from the groupcomprising the sidechains of lysine, histidine, and arginine. In apreferred embodiment, the amino acid side chain is that of lysine.

In a further embodiment, the glycosylated protein is formed using acarbohydrate of molecular weight of at least about 200 Daltons. Inanother embodiment, the glycosylated protein is formed using acarbohydrate of molecular weight of at least about 10000 Daltons. Inanother embodiment, the glycosylated protein is formed using acarbohydrate of molecular weight of at least about 25000 Daltons. Inanother embodiment, the glycosylated protein is formed usingcarbohydrate that is selected from group consisting of monosaccharides,disaccharides, linear oligosaccharides, and branched oligosaccharides asdescribed herein.

In another embodiment, the glycosylated protein is formed using linearoligosaccharides such as chitin, including partially deacetylatedchitin, hyaluronic acid, keratin, keratin sulfate, chondroitin,chondroitin sulfate, dermatan, dermatan sulfate, and heparin, which canachieve molecular weights as much as 5,000,000 Daltons. Preferred sizesof these oligosaccharides include, but are not limited to, those withmolecular weights of at least about 500 Daltons, at least about 1,000Daltons, at least about 1,500 Daltons, at least about 3,500 Daltons, atleast about 5,000 Daltons, at least about 10,000 Daltons, at least about20,000 Daltons, at least about 25,000 Daltons, at least about 33,000Daltons, at least about 50,000 Daltons, and at least about 100,000Daltons. In another embodiment, preferred sizes of theseoligosaccharides include, but are not limited to, those molecularweights that are less than about 6,000 Daltons, and less than about50,000 Daltons.

In another embodiment, the glycosylated protein is formed using branchedoligosaccharides such as high-mannose N-glycans, hybrid N-glycans, andcomplex N-glycans that have molecular weights under 10,000 Daltons.Preferred sizes of these oligosaccharides include, but are not limitedto, those with molecular weights of from about 500 Daltons to about10,000 Daltons. A more preferred size of these oligosaccharides is fromabout 800 Daltons to about 5,000 Daltons. A most preferred size of theseoligosaccharides is from about 800 Daltons to about 4500 Daltons.

Oligosaccharides are available in a range of molecular weights. Linearoligosaccharides based on repeating monosaccharide or disaccharidesubunits, such as hyaluronic acid and the other glycosaminoglycans,²⁷⁻²⁹may be obtained as discreet sized species or a distribution of sizesabout an average molecular weight. Other linear oligosaccharides includechitin, and partially deacetylated chitin.³⁰⁻³¹ The latter refers tochitin that has been processed to remove some or most of the N-acetylmoieties that modify the glucosamine core. The degree of deacetylationrefers to the percentage of N-acetyl moieties removed, with up to 95%removal possible to retain the parent chitin identity. Removal of atleast 49% of the acetyl groups within the polymer molecule is requiredfor solubility in water. A range of molecular weights of the parentchitin and the partially deacetylated chitins are possible.

The molecular weight of a linear oligosaccharide may be obtained from avariety of direct and indirect physical characterization methods. Theseinclude, but are not limited to, high performance liquid chromatography(HPLC), gel permeation chromatography (GPC), mass spectroscopy (MS)including matrix assisted laser desorption ionization mass spectroscopy(MALDI-MS), ultracentrifuge sedimentation, viscosity, osmotic pressure,and dynamic light scattering (DLS), all of which may be affected by thenumber, size, or shape of molecules in a matrix, a suspension, or in asolution.³²⁻³⁵ The molecular weight of high-mannose N-glycans, hybridN-glycans, and complex N-glycans may be determined in similar manner.

For distributions of different sized oligosaccharides, weight-averagemolecular weight (Mw) and molecular weight distributions may bedetermined from ultracentrifuge sedimentation, diffusion, and lightscattering. Number-average molecular weight (Mn) and molecular weightdistributions may be determined from osmotic pressure and intrinsicviscosity determinations. Optical properties are best reflected in theweight-average molecular weight, while strength properties are bestreflected in number-average molecular weight. Mn is the number ofmonomer molecules divided by the total number of molecules times themonomer mole weight. Mw is the area under the weight distribution curvethat is divided into two equal parts. Mn<=Mw.

Weight-average molecular weight, is calculated by the equation

M _(w)={Sum[(W _(i))(MW)_(i)]}/{Sum W _(i)},

where W_(i) is the weight fraction of each size fraction and (MW)_(I) isthe mean molecular weight of the size fraction. The “weight-average”molecular weight is particularly significant in the analysis ofproperties such as viscosity, where the weight of the molecules isimportant. Number-average molecular weight is calculated by the equation

M _(n)={Sum[(W _(i))(MW)_(i)]}/{Sum X _(i)},

where the value X_(i) is the number of molecules in each size fraction.

In another embodiment, the glycosylated protein has one or both of thetautomeric forms

wherein each of R₁, R₂, and R₃ is individually and independentlyselected from the group consisting of H and saccharides defined herein,and wherein R is selected from the group consisting of C1-C6 alkyl,branched C3-C8 alkyl, —(CH₂)_(m)—CN, —(CH₂)_(m)OR6, —(CH₂)_(m)—CO₂H,—(CH₂)_(m)—CO₂R6, —(CH₂)_(m)—NR6(R7), —(CH₂)_(m)—S(O)_(n)—C1-C6 alkyl,—(CH₂)_(m)—C(O)NR6(R7), —(CH₂)_(m)—CO₂—C4-C6 heterocyclyl,—(CH₂)_(m)—C4-C6 heterocyclyl, —(CH₂)_(m)—CO₂—C4-C6 heteroaryl, or—(CH₂)_(m)—C4-C6-heteroaryl, wherein each alkyl may optionally containan ether linkage and, wherein each alkyl is optionally substituted withone or two C1-C6 alkyl groups, each R6 and R7 is individually andindependently H, C1-C6 alkyl, or branched C3-C8 alkyl, each m isindividually and independently 1, 2, 3, 4, or 5, each n is individuallyand independently 0, 1, or 2.

In a preferred embodiment, the glycosylated protein is selected from thegroup consisting of glucose oxidases modified with hyaluronic acid,glucose oxidases modified with chitin, glucose oxidases modified withpartially deacetylated chitin, glucose oxidases modified withhigh-mannose N-glycans, glucose oxidases modified with keratin, glucoseoxidases modified with keratin sulfate, glucose oxidases modified withchondroitin, glucose oxidases modified with chondroitin sulfate, glucoseoxidases modified with dermatan, glucose oxidases modified with dermatansulfate, glucose oxidases modified with heparin, glucose oxidasesmodified with hybrid N-glycans, glucose oxidases modified with complexN-glycans, lactate oxidases modified with hyaluronic acid, lactateoxidases modified with chitin, lactate oxidases modified with partiallydeacetylated chitin, lactate oxidases modified with high-mannoseN-glycans, lactate oxidases modified with keratin, lactate oxidasesmodified with keratin sulfate, lactate oxidases modified withchondroitin, lactate oxidases modified with chondroitin sulfate, lactateoxidases modified with dermatan, lactate oxidases modified with dermatansulfate, lactate oxidases modified with heparin, lactate oxidasesmodified with hybrid N-glycans, lactate oxidases modified with complexN-glycans, alcohol oxidases modified with hyaluronic acid, alcoholoxidases modified with chitin, alcohol oxidases modified with partiallydeacetylated chitin, alcohol oxidases modified with high-mannoseN-glycans, alcohol oxidases modified with keratin, alcohol oxidasesmodified with keratin sulfate, alcohol oxidases modified withchondroitin, alcohol oxidases modified with chondroitin sulfate, alcoholoxidases modified with dermatan, alcohol oxidases modified with dermatansulfate, alcohol oxidases modified with heparin, alcohol oxidasesmodified with hybrid N-glycans, alcohol oxidases modified with complexN-glycans, glutamate oxidases modified with hyaluronic acid, glutamateoxidases modified with chitin, glutamate oxidases modified withpartially deacetylated chitin, glutamate oxidases modified withhigh-mannose N-glycans, glutamate oxidases modified with keratin,glutamate oxidases modified with keratin sulfate, glutamate oxidasesmodified with chondroitin, glutamate oxidases modified with chondroitinsulfate, glutamate oxidases modified with dermatan, glutamate oxidasesmodified with dermatan sulfate, glutamate oxidases modified withheparin, glutamate oxidases modified with hybrid N-glycans, glutamateoxidases modified with complex N-glycans, choline oxidases modified withhyaluronic acid, choline oxidases modified with chitin, choline oxidasesmodified with partially deacetylated chitin, choline oxidases modifiedwith high-mannose N-glycans, choline oxidases modified with keratin,choline oxidases modified with keratin sulfate, choline oxidasesmodified with chondroitin, choline oxidases modified with chondroitinsulfate, choline oxidases modified with dermatan, choline oxidasesmodified with dermatan sulfate, choline oxidases modified with heparin,choline oxidases modified with hybrid N-glycans, choline oxidasesmodified with complex N-glycans, sarcosine oxidases modified withhyaluronic acid, sarcosine oxidases modified with chitin, sarcosineoxidases modified with partially deacetylated chitin, sarcosine oxidasesmodified with high-mannose N-glycans, sarcosine oxidases modified withkeratin, sarcosine oxidases modified with keratin sulfate, sarcosineoxidases modified with chondroitin, sarcosine oxidases modified withchondroitin sulfate, sarcosine oxidases modified with dermatan,sarcosine oxidases modified with dermatan sulfate, sarcosine oxidasesmodified with heparin, sarcosine oxidases modified with hybridN-glycans, sarcosine oxidases modified with complex N-glycans, xanthineoxidases modified with hyaluronic acid, xanthine oxidases modified withchitin, xanthine oxidases modified with partially deacetylated chitin,xanthine oxidases modified with high-mannose N-glycans, xanthineoxidases modified with keratin, xanthine oxidases modified with keratinsulfate, xanthine oxidases modified with chondroitin, xanthine oxidasesmodified with chondroitin sulfate, xanthine oxidases modified withdermatan, xanthine oxidases modified with dermatan sulfate, xanthineoxidases modified with heparin, xanthine oxidases modified with hybridN-glycans, xanthine oxidases modified with complex N-glycans, oxalateoxidases modified with hyaluronic acid, oxalate oxidases modified withchitin, oxalate oxidases modified with partially deacetylated chitin,oxalate oxidases modified with high-mannose N-glycans, oxalate oxidasesmodified with keratin, oxalate oxidases modified with keratin sulfate,oxalate oxidases modified with chondroitin, oxalate oxidases modifiedwith chondroitin sulfate, oxalate oxidases modified with dermatan,oxalate oxidases modified with dermatan sulfate, oxalate oxidasesmodified with heparin, oxalate oxidases modified with hybrid N-glycans,oxalate oxidases modified with complex N-glycans, cholesterol oxidasesmodified with hyaluronic acid, cholesterol oxidases modified withchitin, cholesterol oxidases modified with partially deacetylatedchitin, cholesterol oxidases modified with high-mannose N-glycans,cholesterol oxidases modified with keratin, cholesterol oxidasesmodified with keratin sulfate, cholesterol oxidases modified withchondroitin, cholesterol oxidases modified with chondroitin sulfate,cholesterol oxidases modified with dermatan, cholesterol oxidasesmodified with dermatan sulfate, cholesterol oxidases modified withheparin, cholesterol oxidases modified with hybrid N-glycans,cholesterol oxidases modified with complex N-glycans, histamine oxidasesmodified with hyaluronic acid, histamine oxidases modified with chitin,histamine oxidases modified with partially deacetylated chitin,histamine oxidases modified with high-mannose N-glycans, histamineoxidases modified with keratin, histamine oxidases modified with keratinsulfate, histamine oxidases modified with chondroitin, histamineoxidases modified with chondroitin sulfate, histamine oxidases modifiedwith dermatan, histamine oxidases modified with dermatan sulfate,histamine oxidases modified with heparin, histamine oxidases modifiedwith hybrid N-glycans, histamine oxidases modified with complexN-glycans, glycine oxidases modified with hyaluronic acid, glycineoxidases modified with chitin, glycine oxidases modified with partiallydeacetylated chitin, glycine oxidases modified with high-mannoseN-glycans, glycine oxidases modified with keratin, glycine oxidasesmodified with keratin sulfate, glycine oxidases modified withchondroitin, glycine oxidases modified with chondroitin sulfate, glycineoxidases modified with dermatan, glycine oxidases modified with dermatansulfate, glycine oxidases modified with heparin, glycine oxidasesmodified with hybrid N-glycans, glycine oxidases modified with complexN-glycans, NADH oxidases modified with hyaluronic acid, NADH oxidasesmodified with chitin, NADH oxidases modified with partially deacetylatedchitin, NADH oxidases modified with high-mannose N-glycans, NADHoxidases modified with keratin, NADH oxidases modified with keratinsulfate, NADH oxidases modified with chondroitin, NADH oxidases modifiedwith chondroitin sulfate, NADH oxidases modified with dermatan, NADHoxidases modified with dermatan sulfate, NADH oxidases modified withheparin, NADH oxidases modified with hybrid N-glycans, NADH oxidasesmodified with complex N-glycans, galactose oxidases modified withhyaluronic acid, galactose oxidases modified with chitin, galactoseoxidases modified with partially deacetylated chitin, galactose oxidasesmodified with high-mannose N-glycans, galactose oxidases modified withkeratin, galactose oxidases modified with keratin sulfate, galactoseoxidases modified with chondroitin, galactose oxidases modified withchondroitin sulfate, galactose oxidases modified with dermatan,galactose oxidases modified with dermatan sulfate, galactose oxidasesmodified with heparin, galactose oxidases modified with hybridN-glycans, galactose oxidases modified with complex N-glycans, alcoholdehydrogenases modified with hyaluronic acid, alcohol dehydrogenasesmodified with chitin, alcohol dehydrogenases modified with partiallydeacetylated chitin, alcohol dehydrogenases modified with high-mannoseN-glycans, alcohol dehydrogenases modified with keratin, alcoholdehydrogenases modified with keratin sulfate, alcohol dehydrogenasesmodified with chondroitin, alcohol dehydrogenases modified withchondroitin sulfate, alcohol dehydrogenases modified with dermatan,alcohol dehydrogenases modified with dermatan sulfate, alcoholdehydrogenases modified with heparin, alcohol dehydrogenases modifiedwith hybrid N-glycans, alcohol dehydrogenases modified with complexN-glycans, glucose dehydrogenases modified with hyaluronic acid, glucosedehydrogenases modified with chitin, glucose dehydrogenases modifiedwith partially deacetylated chitin, glucose dehydrogenases modified withhigh-mannose N-glycans, glucose dehydrogenases modified with keratin,glucose dehydrogenases modified with keratin sulfate, glucosedehydrogenases modified with chondroitin, glucose dehydrogenasesmodified with chondroitin sulfate, glucose dehydrogenases modified withdermatan, glucose dehydrogenases modified with dermatan sulfate, glucosedehydrogenases modified with heparin, glucose dehydrogenases modifiedwith hybrid N-glycans, glucose dehydrogenases modified with complexN-glycans, L-lactate dehydrogenases modified with hyaluronic acid,L-lactate dehydrogenases modified with chitin, L-lactate dehydrogenasesmodified with partially deacetylated chitin, L-lactate dehydrogenasesmodified with high-mannose N-glycans, L-lactate dehydrogenases modifiedwith keratin, L-lactate dehydrogenases modified with keratin sulfate,L-lactate dehydrogenases modified with chondroitin, L-lactatedehydrogenases modified with chondroitin sulfate, L-lactatedehydrogenases modified with dermatan, L-lactate dehydrogenases modifiedwith dermatan sulfate, L-lactate dehydrogenases modified with heparin,L-lactate dehydrogenases modified with hybrid N-glycans, L-lactatedehydrogenases modified with complex N-glycans, cortisol dehydrogenasesmodified with hyaluronic acid, cortisol dehydrogenases modified withchitin, cortisol dehydrogenases modified with partially deacetylatedchitin, cortisol dehydrogenases modified with high-mannose N-glycans,cortisol dehydrogenases modified with keratin, cortisol dehydrogenasesmodified with keratin sulfate, cortisol dehydrogenases modified withchondroitin, cortisol dehydrogenases modified with chondroitin sulfate,cortisol dehydrogenases modified with dermatan, cortisol dehydrogenasesmodified with dermatan sulfate, cortisol dehydrogenases modified withheparin, cortisol dehydrogenases modified with hybrid N-glycans,cortisol dehydrogenases modified with complex N-glycans, galactosedehydrogenases modified with hyaluronic acid, galactose dehydrogenasesmodified with chitin, galactose dehydrogenases modified with partiallydeacetylated chitin, galactose dehydrogenases modified with high-mannoseN-glycans, galactose dehydrogenases modified with keratin, galactosedehydrogenases modified with keratin sulfate, galactose dehydrogenasesmodified with chondroitin, galactose dehydrogenases modified withchondroitin sulfate, galactose dehydrogenases modified with dermatan,galactose dehydrogenases modified with dermatan sulfate, galactosedehydrogenases modified with heparin, galactose dehydrogenases modifiedwith hybrid N-glycans, galactose dehydrogenases modified with complexN-glycans, glycerol dehydrogenases modified with hyaluronic acid,glycerol dehydrogenases modified with chitin, glycerol dehydrogenasesmodified with partially deacetylated chitin, glycerol dehydrogenasesmodified with high-mannose N-glycans, glycerol dehydrogenases modifiedwith keratin, glycerol dehydrogenases modified with keratin sulfate,glycerol dehydrogenases modified with chondroitin, glyceroldehydrogenases modified with chondroitin sulfate, glyceroldehydrogenases modified with dermatan, glycerol dehydrogenases modifiedwith dermatan sulfate, glycerol dehydrogenases modified with heparin,glycerol dehydrogenases modified with hybrid N-glycans, glyceroldehydrogenases modified with complex N-glycans, glucose-6-phosphatedehydrogenases modified with hyaluronic acid, glucose-6-phosphatedehydrogenases modified with chitin, glucose-6-phosphate dehydrogenasesmodified with partially deacetylated chitin, glucose-6-phosphatedehydrogenases modified with high-mannose N-glycans, glucose-6-phosphatedehydrogenases modified with keratin, glucose-6-phosphate dehydrogenasesmodified with keratin sulfate, glucose-6-phosphate dehydrogenasesmodified with chondroitin, glucose-6-phosphate dehydrogenases modifiedwith chondroitin sulfate, glucose-6-phosphate dehydrogenases modifiedwith dermatan, glucose-6-phosphate dehydrogenases modified with dermatansulfate, glucose-6-phosphate dehydrogenases modified with heparin,glucose-6-phosphate dehydrogenases modified with hybrid N-glycans,glucose-6-phosphate dehydrogenases modified with complex N-glycans,3-hydroxybutyrate dehydrogenases modified with hyaluronic acid,3-hydroxybutyrate dehydrogenases modified with chitin, 3-hydroxybutyratedehydrogenases modified with partially deacetylated chitin,3-hydroxybutyrate dehydrogenases modified with high-mannose N-glycans,3-hydroxybutyrate dehydrogenases modified with keratin,3-hydroxybutyrate dehydrogenases modified with keratin sulfate,3-hydroxybutyrate dehydrogenases modified with chondroitin,3-hydroxybutyrate dehydrogenases modified with chondroitin sulfate,3-hydroxybutyrate dehydrogenases modified with dermatan,3-hydroxybutyrate dehydrogenases modified with dermatan sulfate,3-hydroxybutyrate dehydrogenases modified with heparin,3-hydroxybutyrate dehydrogenases modified with hybrid N-glycans,3-hydroxybutyrate dehydrogenases modified with complex N-glycans,L-malate dehydrogenases modified with hyaluronic acid, L-malatedehydrogenases modified with chitin, L-malate dehydrogenases modifiedwith partially deacetylated chitin, L-malate dehydrogenases modifiedwith high-mannose N-glycans, L-malate dehydrogenases modified withkeratin, L-malate dehydrogenases modified with keratin sulfate, L-malatedehydrogenases modified with chondroitin, L-malate dehydrogenasesmodified with chondroitin sulfate, L-malate dehydrogenases modified withdermatan, L-malate dehydrogenases modified with dermatan sulfate,L-malate dehydrogenases modified with heparin, L-malate dehydrogenasesmodified with hybrid N-glycans, L-malate dehydrogenases modified withcomplex N-glycans, sorbitol dehydrogenases modified with hyaluronicacid, sorbitol dehydrogenases modified with chitin, sorbitoldehydrogenases modified with partially deacetylated chitin, sorbitoldehydrogenases modified with high-mannose N-glycans, sorbitoldehydrogenases modified with keratin, sorbitol dehydrogenases modifiedwith keratin sulfate, sorbitol dehydrogenases modified with chondroitin,sorbitol dehydrogenases modified with chondroitin sulfate, sorbitoldehydrogenases modified with dermatan, sorbitol dehydrogenases modifiedwith dermatan sulfate, sorbitol dehydrogenases modified with heparin,sorbitol dehydrogenases modified with hybrid N-glycans, and sorbitoldehydrogenases modified with complex N-glycans, and fusion proteinsthereof.

In a more preferred embodiment, the glycosylated protein is selectedfrom the group consisting of glucose oxidases modified with hyaluronicacid, glucose oxidases modified with partially deacetylated chitin,glucose oxidases modified with complex N-glycans, lactate oxidasesmodified with hyaluronic acid, lactate oxidases modified with partiallydeacetylated chitin, lactate oxidases modified with complex N-glycans,alcohol oxidases modified with hyaluronic acid, alcohol oxidasesmodified with partially deacetylated chitin, alcohol oxidases modifiedwith complex N-glycans, glutamate oxidases modified with hyaluronicacid, glutamate oxidases modified with partially deacetylated chitin,glutamate oxidases modified with complex N-glycans, choline oxidasesmodified with hyaluronic acid, choline oxidases modified with partiallydeacetylated chitin, choline oxidases modified with complex N-glycans,sarcosine oxidases modified with hyaluronic acid, sarcosine oxidasesmodified with partially deacetylated chitin, sarcosine oxidases modifiedwith complex N-glycans, xanthine oxidases modified with hyaluronic acid,xanthine oxidases modified with partially deacetylated chitin, xanthineoxidases modified with complex N-glycans, cholesterol oxidases modifiedwith hyaluronic acid, cholesterol oxidases modified with partiallydeacetylated chitin, cholesterol oxidases modified with complexN-glycans, histamine oxidases modified with hyaluronic acid, histamineoxidases modified with partially deacetylated chitin, histamine oxidasesmodified with complex N-glycans, NADH oxidases modified with hyaluronicacid, NADH oxidases modified with partially deacetylated chitin, NADHoxidases modified with complex N-glycans, alcohol dehydrogenasesmodified with hyaluronic acid, alcohol dehydrogenases modified withpartially deacetylated chitin, alcohol dehydrogenases modified withcomplex N-glycans, glucose dehydrogenases modified with hyaluronic acid,glucose dehydrogenases modified with partially deacetylated chitin,glucose dehydrogenases modified with complex N-glycans, L-lactatedehydrogenases modified with hyaluronic acid, L-lactate dehydrogenasesmodified with partially deacetylated chitin, L-lactate dehydrogenasesmodified with complex N-glycans, cortisol dehydrogenases modified withhyaluronic acid, cortisol dehydrogenases modified with partiallydeacetylated chitin, cortisol dehydrogenases modified with complexN-glycans, L-malate dehydrogenases modified with hyaluronic acid,L-malate dehydrogenases modified with partially deacetylated chitin, andL-malate dehydrogenases modified with complex N-glycans, and fusionproteins thereof.

In another aspect of the present invention, the glycosylated protein,fabricated by the methods disclosed herein, is used in the assembly ofan amperometric biosensor. The amperometric biosensor comprises acounter electrode, a reference electrode, one or more optional rejectionlayers, and a working electrode that comprises a sensing element, whichcomprises a support having a surface; and a layer on the surface,wherein the layer comprises an amino sidechain glycosylated enzyme,wherein the enzyme is predominantly in its active form. The counter andreference electrodes may be separate to form a three-electrodeamperometric biosensor, or combined to form a two-electrode amperometricbiosensor. The layer on the surface containing the amino sidechainglycosylated enzyme may optionally contain other proteins andcrosslinking agents.

FIG. 21 is a schematic drawing of a prototypical working electrode in anamperometric biosensor. 101 and 103 are optional rejection layers, whichmay have the same or different compositions. 102 is the layer where theamino sidechain glycosylated enzyme and other optional proteins andcrosslinking agents reside. 104 is the working electrode sensing elementand supports 102 and 103. 104 is typically held at a bias of 0.4 to 0.9V versus a Ag/AgCl reference electrode. 105 is the current generated at104 by virtue of the peroxide generated by the enzyme in 102. One ofordinary skill in the art will recognize that the sensing element 106would be the same regardless of whether the biosensor employs a twoelectrode configuration (wherein the reference and counter electrode arecombined), or a three electrode configuration (wherein the reference andcounter electrodes are separate entities).

In a preferred embodiment, the glycosylated protein is an enzymeindependently selected from the group consisting of glucose oxidases,lactate oxidases, alcohol oxidases, glutamate oxidases, xanthineoxidases, sarcosine oxidases, cholesterol oxidases, oxalate oxidases,D-amino acid oxidases, choline oxidases, glutathione sulfhydryloxidases, (S)-6-hydroxynicotine oxidases, (R)-6-hydroxynicotineoxidases, pyruvate oxidases, acyl coenzyme A oxidases, glycerolphosphateoxidases, GABA oxidases, histamine oxidases, diamine oxidases,nucleoside oxidases, L-lysine oxidases, L-aspartate oxidases, glycineoxidases, galactose oxidases, NADH oxidases, glucose dehydrogenases,alcohol dehydrogenases, cortisol dehydrogenases, lactate dehydrogenases,and fusion proteins thereof.

In another preferred embodiment, the glycosylated protein isglycosylated with a carbohydrate that is selected from the groupconsisting of chitin, partially deacylated chitin, hyaluronic acid,keratin, keratin sulfate, chondroitin, chondroitin sulfate, dermatan,dermatan sulfate, heparin, native N-glycan cores, high-mannoseN-glycans, hybrid N-glycans, complex N-glycans, and derivatives thereof.

In another embodiment of the present invention, the glycosylatedproteins may find use in human health monitoring applications. Inanother embodiment of the present invention, the glycosylated proteinsmay find use in non-human mammal health monitoring applications.

The following examples set forth preferred methods in accordance withthe invention. It is understood, however, that these examples areprovided by way of illustration and nothing therein should be taken as alimitation upon the overall scope of the invention.

Protein expression and purification: Proteins glucose oxidase (GOx),MBP-glucose oxidase (MBP-GOx), lactate oxidase (LOx), MBP-lactateoxidase (MBP-LOx), MBP-putrescene oxidase (MBP-PutOx), glycine oxidase(GlyOx), sarcosine oxidase (SOx), glutamate oxidase (GlutOx), alcoholoxidase (AOx), NctB oxidase (NicOx), cholesterol oxidase fromBrevibacterium (COx2), MBP-cholesterol oxidase from sp. Penicillium(MBP-COx1), alcohol dehydrogenase (ADH), cortisol dehydrogenase (CDH),glucose dehydrogenase (GDH), L-lactate dehydrogenase (LDH), malatedehydrogenase (MDH)] were either purchased from commercial vendors orprepared by standard recombinant over-expression techniques usingpublished methods³⁶ in E. coli. ³⁷ or Pichia Pastoris ³⁸⁻³⁹, andpurified by standard methods.⁴⁰

Carbohydrates: N-acetylglucosamine (GlcNAc), N-acetylmannosamine(ManNAc), N-acetylgalactosamine (GalNAc), diacetylchitobiose (DACB),triacetylchitotriose (TACT), Lewis B tetrasaccharide (LewB4), nativeN-glycan cores Man-3 and Man-5, and hyaluronic acid polymers of variouslengths including those of less than 6K Mw (HA6), and those of less than50K Mw (HA50) were obtained from commercial sources.N-propanylglucosamine, N-n-butanylglucosamine, N-isopropanylglucosamine,N-2,2,dimethylpropanylglucosamine, N-((2-oxoethoxy)-propanyl)glucosaminewere synthesized using the method of Inouye et al.⁴¹

General oxazoline formation protocol: A carbohydrate with anN-acyl-2-amino moiety at the reducing end was dissolved in deionizedH₂O. At least 7.5 molar equivalents of triethylamine or Na₃PO₄ wereadded until the pH was 11. Three molar equivalents of either DMC orCDMBI were added and the reaction incubated at 4° C. from 2 to 24 hours.The pH of the solution was then adjusted to pH 8-8.5 with 1 M bicine orto pH 6, 7, or 8 with 1M MES. After adjustment of pH, some oxazolineswere flash frozen in liquid nitrogen and stored at −80° C. until use.CDMBI (2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride) wasprepared according to the method of Noguchi et al.²¹ DMC(chloro-1,3-dimethylimidazolinium chloride) was purchased fromcommercial sources.

General preparation of protein: Protein was prepared for conjugation byeither dissolving lyophilized protein in PBS, pH 8.0 followed bydesalting via a G-50 spin column equilibrated with PBS pH 8.0, ordissolved in PBS, pH 8.0, and used directly. The protein concentrationwas determined by triplicate Bradford assay.⁴² Of the solution, thevolume of solution containing from about 5 ug to 50 mg was aliquotedinto a microfuge tube for use in the conjugation reaction.

General conjugation of neutralized oxazoline carbohydrate with aprotein: The neutralized oxazolines and target proteins were prepared asabove. The protein solution was dispensed into a microfuge tube followedby addition of about a 100 to 1000-fold molar excess of the carbohydrateoxazoline and allowed to react from 2 hours to 72 hours at 4° C. or roomtemperature. Up to 5 ug samples were withdrawn for SDS-PAGE gel mobilityshift assay to determine reaction completeness. Novex 8% and/or 12%bis-tris SDS-PAGE gels were used with a MES-SDS running buffer. Theremainder of the samples were then cryopreserved by addition of glycerolto 20% v/v, flash frozen in liquid nitrogen, and stored at −80° C.

SDS-PAGE gels were stained with PageBlue (Thermo Scientific) anddestained in deionized water optionally supplemented with 1% aceticacid.³⁷ Destained gels were visualized on an optical trans-illuminator,and the relative gel mobility evaluated based on the lagging edge ofeach band. Conjugated constructs derived from highly chargedcarbohydrate oxazolines (i.e. hyaluronic acid, the glycosaminoglycans,partially deacetylated chitin)²⁹ may produce distortion in the bandmorphology, wherein the band morphology being manifest as distortion,streaking, smearing, lengthening, lightening of the stained band,formation of a chevron-shaped and/or U-shaped band, and/or extension ofthe band wherein the distal portions of the band display retardedmobility relative to the center of the band in the lane, and wherein thechange in morphology is relative to the unconjugated protein sampleband.^(37, 43-45)

Enzymatic activity assay: Samples were thawed for assay and desaltedinto PBS (140 mM NaCl, 10 mM NaPO₄ pH 7.4) via Sephadex G-50 spincolumn. The concentration of each desalted protein sample was determinedby triplicate Bradford assay using previously described methods.⁴²Oxidase enzymes were assayed in triplicate by Amplex red peroxidasecoupled assay @ RT under saturating substrate conditions usingpreviously described methods.⁴⁶⁻⁴⁷ Dehydrogenase enzymes were assayed intriplicate by direct spectrophotometric detection of the NADH product atroom temperature under saturating substrate concentrations, usingpreviously described methods.⁴⁸ Enzyme concentrations giving opticalrates between 0.01 ΔA*min-1 and 0.2 ΔA*min-1 were employed for allenzymes. Mass specific activity (pmoles product*min-1*mg-1, U/mg) ofeach control sample and their conjugates were calculated using theexperimentally determined slope of either the hydrogen peroxide standardcurve (0.0308 A560*cm-1*μM-1) or the μM extinction coefficient of NADH(0.006220 A340*cm-1*μM-1), the optical rate of each assay (ΔA560*min-1),and the mass of enzyme of enzyme in mg in the assay. Data was plotted inPrism software (GraphPad).

Water bath activity study of glycan-protein conjugates: After assay,protein samples were incubated in a water bath @ 42° C. for 24-96 hours,incubated on ice for 2 minutes, then centrifuged for 10 s @ 10,000 rpm.Determination of the mass specific activity of these samples was thenperformed as described in previously. Specific activity values beforeand after thermal incubation were plotted with Prism software(GraphPad).

Fluorescence thermal shift assay (FTSA) of glycan-protein conjugates:The impact of carbohydrate conjugation on the thermal stability wasevaluated by a SYPRO-Orange dye association fluorescence thermal shiftassay.⁴⁹ Melts were performed on each control protein and each of itsrespective conjugates. After activity assay, protein samples wereprepared for fluorescence thermal shift assay by dilution to 0.1-5.0 μMin a 2 mL final volume with PBS supplemented with 2.5× SYPRO-Orange dye(Invitrogen). Fluorescence emission intensity was observed vstemperature in a Cary Eclipse fluorescence spectrophotometer equippedwith a multi cell peltier (484 nm ex, 620 nm em, 1 sec integration time,10 nm em/ex slit width, 1° C./minute temperature ramp rate, 0.5 C datainterval, medium detector sensitivity). Data was collected from 20° C.to 85° C. or until the fluorescence intensity began to decline from itsmaximum. The fluorescence intensity values were normalized relative tothe initial and maximum observed values and the apparent Tm determinedby fitting the resulting data to a two state Boltzmann sigmoid bynonlinear regression analysis using Prism software (GraphPad).

The representative SDS-PAGE gel image in FIG. 1 demonstrates, by virtueof the observed band shifts between the native MDH enzyme and theglycosylated MDH enzyme, that coupling a nucleophilic amino sidechainoccurs and is independent of the stereochemistry within the oxazolinering (Examples 1, 2, and 3).

The representative SDS-PAGE gel image in FIG. 2 demonstrates, by virtueof the observed band shifts between the native MDH and LDH enzymes andthe glycosylated forms thereof, that the coupling of both small andlarge linear oligosaccharides occurs and is independent of the size ofthe linear oligosaccharide (Examples 4, 7, 26, and 28).

The representative SDS-PAGE gel image in FIG. 3 demonstrates, by virtueof the observed band shifts between the native LOx and SOx enzymes andthe glycosylated forms thereof, that the coupling of both small andlarge linear oligosaccharides occurs and is independent of the size ofthe linear oligosaccharide (Examples 58, 60, 72, and 74).

The representative SDS-PAGE gel image in FIG. 4 demonstrates, by virtueof the observed band shifts between the native CDH and ADH enzymes andthe glycosylated forms thereof, that the coupling of both small andlarge linear oligosaccharides occurs and is independent of the size ofthe linear oligosaccharide (Examples 14, 16, 18, and 20).

The representative SDS-PAGE gel image in FIG. 5 demonstrates, by virtueof the observed band shifts between native AOx enzyme and theglycosylated forms thereof, that the coupling of both small and largelinear oligosaccharides occurs and is independent of the size of thelinear oligosaccharide (Examples 30 and 32).

The representative SDS-PAGE gel image in FIG. 6 demonstrates, by virtueof the observed band shifts between the native MDH and SOx enzymes andthose glycosylated with LewB4, that the coupling of a fucosyl complexN-glycan oligosaccharides occurs and is independent of enzyme type(Examples 5 and 61).

The representative SDS-PAGE gel image in FIG. 7 demonstrates, by virtueof the observed band shifts between the native MDH enzyme and theglycosylated MDH enzyme, that coupling a nucleophilic amino sidechainoccurs and is independent of the R moiety on the oxazoline ring(Examples 8, 9, 10, 11, and 12).

The representative SDS-PAGE gel image in FIG. 8 demonstrates, by virtueof the observed band shifts between the native SOx enzyme and thatglycosylated with Man-3 and Man-5, that the coupling of N-glycan coresand high-mannose N-glycans oligosaccharides occurs and is independent ofglycan type (Examples 62 and 63).

The representative SDS-PAGE gel image in FIG. 9 demonstrates, by virtueof the observed band shifts between the native SOx enzymes and the TACTglycosylated form, that the coupling occurs over a range of pH values,including those that are slightly acidic (Examples 58, 77, and 78).

The representative triplicate mass specific activity determinations inFIG. 10 demonstrate, by virtue of the observed activity, that the nativeGOx enzyme substantially retains activity after glycosylation with arange of different carbohydrates (monosaccharide, trisaccharide, andlinear oligosaccharides), thereby demonstrating that glycosylation doesnot substantially alter either structure or function (Examples 64, 65,66, and 67). These data further demonstrate that GOx can behyper-glycosylated, as the native enzyme naturally possesses one or moreN-glycan units in the traditional sense.

The representative triplicate mass specific activity determinations inFIG. 11 demonstrate, by virtue of the observed activity, that the nativeLOx enzyme substantially retains activity after glycosylation with arange of different carbohydrates (monosaccharide, trisaccharide, andlinear oligosaccharides), thereby demonstrating that glycosylation doesnot substantially alter either structure or function (Examples 70, 72,73, and 74).

The representative triplicate mass specific activity determinations inFIG. 12 demonstrate, by virtue of the observed activity, that the nativeNicOx enzyme substantially retains activity after glycosylation with arange of different carbohydrates (monosaccharide, trisaccharide, andlinear oligosaccharides), thereby demonstrating that glycosylation doesnot substantially alter either structure or function (Examples 49, 50,51, and 52).

The representative triplicate mass specific activity determinations inFIG. 13 demonstrate, by virtue of the observed activity, that the nativeAOx enzyme substantially retains activity after glycosylation with arange of different carbohydrates (monosaccharide and trisaccharide),thereby demonstrating that in many cases glycosylation does notsubstantially alter either structure or function (Examples 29, 30, 31,and 32).

The representative triplicate mass specific activity determinations inFIG. 14 demonstrate, by virtue of the observed activity, that the nativeCortDH enzyme substantially retains activity after glycosylation with arange of different carbohydrates (monosaccharide, trisaccharide, andlinear oligosaccharides), thereby demonstrating that glycosylation doesnot substantially alter either structure or function (Examples 79, 80,81, and 82).

The representative triplicate mass specific activity determinations inFIG. 15 demonstrate, by virtue of the observed activity, that the nativeLDH enzyme substantially retains activity after glycosylation with arange of different carbohydrates (monosaccharide, trisaccharide, andlinear oligosaccharides), thereby demonstrating that glycosylation doesnot substantially alter either structure or function (Examples 25, 26,27, and 28).

The representative triplicate mass specific activity determinations inFIG. 16 demonstrate, by virtue of the observed activity, that the nativeADH enzyme substantially retains activity after glycosylation with arange of different carbohydrates (trisaccharide, and linearoligosaccharides), thereby demonstrating that glycosylation in mostcases does not substantially alter either structure or function(Examples 13, 14, 15, and 16).

The representative triplicate mass specific activity determinations inFIG. 17 demonstrate, by virtue of the observed activity, that nativeGOx, when glycosylated using the current invention, substantiallyretains activity when heated to 42° C. for 96 hours (Examples 64, 65,66, and 67). These data further demonstrate that GOx can behyper-glycosylated, as the native enzyme naturally possesses one or moreN-glycan units in the traditional sense, and that the different glycansused for hyper-glycosylation may substantially improve the stability ofthe native GOx enzyme.

The representative FTSA comparisons in FIG. 18 demonstrate, by virtue ofthe observed shift in fluorescence activity, that native GOx, whenglycosylated using the current invention, shows an improvement inthermal stability (Examples 64, 65, and 66).

The representative FTSA comparisons in FIG. 19 demonstrate, by virtue ofthe observed shift in fluorescence activity, that native LOx, whenglycosylated using the current invention, shows an improvement inthermal stability (Examples 70, 72, 73, and 74).

Example 1: In a manner similar to that described above, GlcNAc wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with MDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the GlcNAc oxazoline occurred.

Example 2: In a manner similar to that described above, ManNAc wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with MDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the ManNAc oxazoline occurred.

Example 3: In a manner similar to that described above, GalNAc wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with MDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the GalNAc oxazoline occurred.

Example 4: In a manner similar to that described above, TACT wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with MDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the TACT oxazoline occurred.

Example 5: In a manner similar to that described above, LewB4 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with MDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the LewB4 oxazoline occurred.

Example 6: In a manner similar to that described above, HA6 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with MDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA6 oxazoline occurred.

Example 7: In a manner similar to that described above, HA50 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with MDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA50 oxazoline occurred.

Example 8: In a manner similar to that described above,N-propanylglucosamine was converted to the corresponding carbohydrateoxazoline with CDMBI, which was subsequently reacted with MDH for about16 hours at room temperature. SDS-PAGE electrophoresis of the resultingmodified protein showed a discernible shift indicating thatglycosylation of the native protein by the N-propanylglucosamineoxazoline occurred.

Example 9: In a manner similar to that described above,N-n-butanylglucosamine was converted to the corresponding carbohydrateoxazoline with CDMBI, which was subsequently reacted with MDH for about16 hours at room temperature. SDS-PAGE electrophoresis of the resultingmodified protein showed a discernible shift indicating thatglycosylation of the native protein by the N-n-butanylglucosamineoxazoline occurred.

Example 10: In a manner similar to that described above,N-isopropanylglucosamine was converted to the corresponding carbohydrateoxazoline with CDMBI, which was subsequently reacted with MDH for about16 hours at room temperature. SDS-PAGE electrophoresis of the resultingmodified protein showed a discernible shift indicating thatglycosylation of the native protein by the N-isopropanylglucosamineoxazoline occurred.

Example 11: In a manner similar to that described above,N-2,2,dimethylpropanylglucosamine was converted to the correspondingcarbohydrate oxazoline with CDMBI, which was subsequently reacted withMDH for about 16 hours at room temperature. SDS-PAGE electrophoresis ofthe resulting modified protein showed a discernible shift indicatingthat glycosylation of the native protein by theN-2,2,dimethylpropanylglucosamine oxazoline occurred.

Example 12: In a manner similar to that described above,N-((2-oxoethoxy)-propanyl)glucosamine was converted to the correspondingcarbohydrate oxazoline with CDMBI, which was subsequently reacted withMDH for about 16 hours at room temperature. SDS-PAGE electrophoresis ofthe resulting modified protein showed a discernible shift indicatingthat glycosylation of the native protein by theN-((2-oxoethoxy)-propanyl)glucosamine oxazoline occurred.

Example 13: In a manner similar to that described above, GlcNAc wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with ADH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the GlcNAc oxazoline occurred. The glycosylated ADH wasdesalted and assayed for enzymatic activity as described above.

Example 14: In a manner similar to that described above, TACT wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with ADH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the TACT oxazoline occurred. The glycosylated ADH wasdesalted and assayed for enzymatic activity as described above.

Example 15: In a manner similar to that described above, HA6 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with ADH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA6 oxazoline occurred. The glycosylated ADH was desaltedand assayed for enzymatic activity as described above.

Example 16: In a manner similar to that described above, HA50 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with ADH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA50 oxazoline occurred. The glycosylated ADH wasdesalted and assayed for enzymatic activity as described above.

Example 17: In a manner similar to that described above, GlcNAc wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with CDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the GlcNAc oxazoline occurred. The glycosylated ADH wasdesalted and assayed for enzymatic activity as described above.

Example 18: In a manner similar to that described above, TACT wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with CDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the TACT oxazoline occurred. The glycosylated ADH wasdesalted and assayed for enzymatic activity as described above.

Example 19: In a manner similar to that described above, HA6 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with CDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA6 oxazoline occurred. The glycosylated ADH was desaltedand assayed for enzymatic activity as described above.

Example 20: In a manner similar to that described above, HA50 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with CDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA50 oxazoline occurred. The glycosylated ADH wasdesalted and assayed for enzymatic activity as described above.

Example 21: In a manner similar to that described above, GlcNAc wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with GDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the GlcNAc oxazoline occurred.

Example 22: In a manner similar to that described above, TACT wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with GDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the TACT oxazoline occurred.

Example 23: In a manner similar to that described above, HA6 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with GDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA6 oxazoline occurred.

Example 24: In a manner similar to that described above, HA50 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with GDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA50 oxazoline occurred.

Example 25: In a manner similar to that described above, GlcNAc wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with LDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the GlcNAc oxazoline occurred. The glycosylated LDH wasdesalted and assayed for enzymatic activity as described above.

Example 26: In a manner similar to that described above, TACT wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with LDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the TACT oxazoline occurred. The glycosylated LDH wasdesalted and assayed for enzymatic activity as described above.

Example 27: In a manner similar to that described above, HA6 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with LDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA6 oxazoline occurred. The glycosylated LDH was desaltedand assayed for enzymatic activity as described above.

Example 28: In a manner similar to that described above, HA50 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with LDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA50 oxazoline occurred. The glycosylated LDH wasdesalted and assayed for enzymatic activity as described above.

Example 29: In a manner similar to that described above, GlcNAc wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with AOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the GlcNAc oxazoline occurred. The glycosylated AOx wasdesalted and assayed for enzymatic activity as described above.

Example 30: In a manner similar to that described above, TACT wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with AOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the TACT oxazoline occurred. The glycosylated AOx wasdesalted and assayed for enzymatic activity as described above.

Example 31: In a manner similar to that described above, HA6 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with AOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA6 oxazoline occurred. The glycosylated AOx was desaltedand assayed for enzymatic activity as described above.

Example 32: In a manner similar to that described above, HA50 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with AOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA50 oxazoline occurred. The glycosylated AOx wasdesalted and assayed for enzymatic activity as described above.

Example 33: In a manner similar to that described above, GlcNAc wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with MBP-COx1 for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the GlcNAc oxazoline occurred. The glycosylated MBP-COx1 wasdesalted and assayed for enzymatic activity as described above.

Example 34: In a manner similar to that described above, TACT wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with MBP-COx1 for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the TACT oxazoline occurred. The glycosylated MBP-COx1 wasdesalted and assayed for enzymatic activity as described above.

Example 35: In a manner similar to that described above, HA6 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with MBP-COx1 for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA6 oxazoline occurred. The glycosylated MBP-COx1 wasdesalted and assayed for enzymatic activity as described above.

Example 36: In a manner similar to that described above, HA50 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with MBP-COx1 for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA50 oxazoline occurred. The glycosylated MBP-COx1 wasdesalted and assayed for enzymatic activity as described above.

Example 37: In a manner similar to that described above, GlcNAc wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with COx2 for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the GlcNAc oxazoline occurred. The glycosylated COx2 wasdesalted and assayed for enzymatic activity as described above.

Example 38: In a manner similar to that described above, TACT wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with COx2 for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the TACT oxazoline occurred. The glycosylated COx2 wasdesalted and assayed for enzymatic activity as described above.

Example 39: In a manner similar to that described above, HA6 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with COx2 for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA6 oxazoline occurred. The glycosylated COx2 wasdesalted and assayed for enzymatic activity as described above.

Example 40: In a manner similar to that described above, HA50 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with COx2 for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA50 oxazoline occurred. The glycosylated COx2 wasdesalted and assayed for enzymatic activity as described above.

Example 41: In a manner similar to that described above, GlcNAc wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with GlutOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the GlcNAc oxazoline occurred. The glycosylated GlutOx wasdesalted and assayed for enzymatic activity as described above.

Example 42: In a manner similar to that described above, TACT wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with GlutOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the TACT oxazoline occurred. The glycosylated GlutOx wasdesalted and assayed for enzymatic activity as described above.

Example 43: In a manner similar to that described above, HA6 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with GlutOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA6 oxazoline occurred. The glycosylated GlutOx wasdesalted and assayed for enzymatic activity as described above.

Example 44: In a manner similar to that described above, HA50 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with GlutOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA50 oxazoline occurred. The glycosylated GlutOx wasdesalted and assayed for enzymatic activity as described above.

Example 45: In a manner similar to that described above, GlcNAc wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with GlyOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the GlcNAc oxazoline occurred. The glycosylated GlyOx wasdesalted and assayed for enzymatic activity as described above.

Example 46: In a manner similar to that described above, TACT wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with GlyOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the TACT oxazoline occurred. The glycosylated GlyOx wasdesalted and assayed for enzymatic activity as described above.

Example 47: In a manner similar to that described above, HA6 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with GlyOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA6 oxazoline occurred. The glycosylated GlyOx wasdesalted and assayed for enzymatic activity as described above.

Example 48: In a manner similar to that described above, HA50 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with GlyOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA50 oxazoline occurred. The glycosylated GlyOx wasdesalted and assayed for enzymatic activity as described above.

Example 49: In a manner similar to that described above, GlcNAc wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with NicOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the GlcNAc oxazoline occurred. The glycosylated NicOx wasdesalted and assayed for enzymatic activity as described above.

Example 50: In a manner similar to that described above, TACT wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with NicOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the TACT oxazoline occurred. The glycosylated NicOx wasdesalted and assayed for enzymatic activity as described above.

Example 51: In a manner similar to that described above, HA6 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with NicOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA6 oxazoline occurred. The glycosylated NicOx wasdesalted and assayed for enzymatic activity as described above.

Example 52: In a manner similar to that described above, HA50 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with NicOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA50 oxazoline occurred. The glycosylated NicOx wasdesalted and assayed for enzymatic activity as described above.

Example 53: In a manner similar to that described above, GlcNAc wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with MBP-PutOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the GlcNAc oxazoline occurred. The glycosylated MBP-PutOx wasdesalted and assayed for enzymatic activity as described above.

Example 54: In a manner similar to that described above, TACT wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with MBP-PutOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the TACT oxazoline occurred. The glycosylated MBP-PutOx wasdesalted and assayed for enzymatic activity as described above.

Example 55: In a manner similar to that described above, HA6 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with MBP-PutOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA6 oxazoline occurred. The glycosylated MBP-PutOx wasdesalted and assayed for enzymatic activity as described above.

Example 56: In a manner similar to that described above, HA50 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with MBP-PutOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA50 oxazoline occurred. The glycosylated MBP-PutOx wasdesalted and assayed for enzymatic activity as described above.

Example 57: In a manner similar to that described above, GlcNAc wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with SOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the GlcNAc oxazoline occurred. The glycosylated SOx wasdesalted and assayed for enzymatic activity as described above.

Example 58: In a manner similar to that described above, TACT wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with SOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the TACT oxazoline occurred. The glycosylated SOx wasdesalted and assayed for enzymatic activity as described above.

Example 59: In a manner similar to that described above, HA6 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with SOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA6 oxazoline occurred. The glycosylated SOx was desaltedand assayed for enzymatic activity as described above.

Example 60: In a manner similar to that described above, HA50 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with SOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA50 oxazoline occurred. The glycosylated SOx wasdesalted and assayed for enzymatic activity as described above.

Example 61: In a manner similar to that described above, LewB4 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with SOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the LewB4 oxazoline occurred. The glycosylated SOx wasdesalted and assayed for enzymatic activity as described above.

Example 62: In a manner similar to that described above, Man-3 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with SOx for about 48-72 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the Man-3 oxazoline occurred. The glycosylated SOx wasdesalted and assayed for enzymatic activity as described above.

Example 63: In a manner similar to that described above, Man-5 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with SOx for about 48-72 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the Man-5 oxazoline occurred. The glycosylated SOx wasdesalted and assayed for enzymatic activity as described above.

Example 64: In a manner similar to that described above, GlcNAc wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with GOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the GlcNAc oxazoline occurred. The glycosylated GOx wasdesalted and assayed for enzymatic activity as described above.

Example 65: In a manner similar to that described above, TACT wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with GOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the TACT oxazoline occurred. The glycosylated GOx wasdesalted and assayed for enzymatic activity as described above.

Example 66: In a manner similar to that described above, HA6 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with GOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA6 oxazoline occurred. The glycosylated GOx was desaltedand assayed for enzymatic activity as described above.

Example 67: In a manner similar to that described above, HA50 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with GOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA50 oxazoline occurred. The glycosylated GOx wasdesalted and assayed for enzymatic activity as described above.

Example 68: In a manner similar to that described above, GlcNAc wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with MBP-GOx for about 72 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the GlcNAc oxazoline occurred. The glycosylated MBP-GOx wasdesalted and assayed for enzymatic activity as described above.

Example 69: In a manner similar to that described above, TACT wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with MBP-GOx for about 72 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the TACT oxazoline occurred. The glycosylated MBP-GOx wasdesalted and assayed for enzymatic activity as described above.

Example 70: In a manner similar to that described above, GlcNAc wasconverted to the corresponding carbohydrate oxazoline with either DMC orCDMBI, which was subsequently reacted with LOx for about 16 hours atroom temperature. SDS-PAGE electrophoresis of the resulting modifiedprotein showed a discernible shift indicating that glycosylation of thenative protein by the GlcNAc oxazoline occurred. The glycosylated LOxwas desalted and assayed for enzymatic activity as described above.

Example 71: In a manner similar to that described above, DACB wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with LOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the DACB oxazoline occurred. The glycosylated LOx wasdesalted and assayed for enzymatic activity as described above.

Example 72: In a manner similar to that described above, TACT wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with LOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the TACT oxazoline occurred. The glycosylated LOx wasdesalted and assayed for enzymatic activity as described above.

Example 73: In a manner similar to that described above, HA6 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with LOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA6 oxazoline occurred. The glycosylated LOx was desaltedand assayed for enzymatic activity as described above.

Example 74: In a manner similar to that described above, HA50 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with LOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA50 oxazoline occurred. The glycosylated LOx wasdesalted and assayed for enzymatic activity as described above.

Example 75: In a manner similar to that described above, GlcNAc wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with MBP-LOx for about 72 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the GlcNAc oxazoline occurred. The glycosylated MBP-LOx wasdesalted and assayed for enzymatic activity as described above.

Example 76: In a manner similar to that described above, TACT wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with MBP-LOx for about 72 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the TACT oxazoline occurred. The glycosylated MBP-LOx wasdesalted and assayed for enzymatic activity as described above.

Example 77: In a manner similar to that described above, TACT wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with SOx at a pH of 6 for about 16 hours atroom temperature. SDS-PAGE electrophoresis of the resulting modifiedprotein showed a discernible shift indicating that glycosylation of thenative protein by the TACT oxazoline occurred.

Example 78: In a manner similar to that described above, TACT wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with SOx at a pH of 7 for about 16 hours atroom temperature. SDS-PAGE electrophoresis of the resulting modifiedprotein showed a discernible shift indicating that glycosylation of thenative protein by the TACT oxazoline occurred.

Example 79: In a manner similar to that described above, GlcNAc wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with CortDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the GlcNAc oxazoline occurred. The glycosylated CortDH wasdesalted and assayed for enzymatic activity as described above.

Example 80: In a manner similar to that described above, TACT wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with CortDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the TACT oxazoline occurred. The glycosylated CortDH wasdesalted and assayed for enzymatic activity as described above.

Example 81: In a manner similar to that described above, HA6 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with CortDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA6 oxazoline occurred. The glycosylated CortDH wasdesalted and assayed for enzymatic activity as described above.

Example 82: In a manner similar to that described above, HA50 wasconverted to the corresponding carbohydrate oxazoline with CDMBI, whichwas subsequently reacted with CortDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshowed a discernible shift indicating that glycosylation of the nativeprotein by the HA50 oxazoline occurred. The glycosylated CortDH wasdesalted and assayed for enzymatic activity as described above.

Example 83: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with GOx for about 16 hours at room temperature.SDS-PAGE electrophoresis of the resulting modified protein shows adiscernible shift indicating that glycosylation of the native protein bythe partially deacetylated chitin oxazoline occurs. The glycosylated GOxis desalted and assayed for enzymatic activity as described above.

Example 84: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with MBP-GOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by the partially deacetylated chitin oxazoline occurs. Theglycosylated MBP-GOx is desalted and assayed for enzymatic activity asdescribed above.

Example 85: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with LOx for about 16 hours at room temperature.SDS-PAGE electrophoresis of the resulting modified protein shows adiscernible shift indicating that glycosylation of the native protein bythe partially deacetylated chitin oxazoline occurs. The glycosylated LOxis desalted and assayed for enzymatic activity as described above.

Example 86: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with MBP-LOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by the partially deacetylated chitin oxazoline occurs. Theglycosylated MBP-LOx is desalted and assayed for enzymatic activity asdescribed above.

Example 87: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with AOx for about 16 hours at room temperature.SDS-PAGE electrophoresis of the resulting modified protein shows adiscernible shift indicating that glycosylation of the native protein bythe partially deacetylated chitin oxazoline occurs. The glycosylated AOxis desalted and assayed for enzymatic activity as described above.

Example 88: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with MBP-AOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by the partially deacetylated chitin oxazoline occurs. Theglycosylated MBP-AOx is desalted and assayed for enzymatic activity asdescribed above.

Example 89: In the following hypothetical example and in a mannersimilar to that described above, HA50 is converted to the correspondingcarbohydrate oxazoline with CDMBI, which is subsequently reacted withMBP-AOx for about 16 hours at room temperature. SDS-PAGE electrophoresisof the resulting modified protein shows a discernible shift indicatingthat glycosylation of the native protein by the HA50 oxazoline occurs.The glycosylated MBP-AOx is desalted and assayed for enzymatic activityas described above.

Example 90: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with GlutOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by the partially deacetylated chitin oxazoline occurs. Theglycosylated GlutOx is desalted and assayed for enzymatic activity asdescribed above.

Example 91: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with MBP-GlutOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by the partially deacetylated chitin oxazoline occurs. Theglycosylated MBP-GlutOx is desalted and assayed for enzymatic activityas described above.

Example 92: In the following hypothetical example and in a mannersimilar to that described above, HA50 is converted to the correspondingcarbohydrate oxazoline with CDMBI, which is subsequently reacted withMBP-GlutOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by the HA50oxazoline occurs. The glycosylated MBP-GlutOx is desalted and assayedfor enzymatic activity as described above.

Example 93: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with choline oxidase for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by the partially deacetylated chitin oxazoline occurs. Theglycosylated choline oxidase is desalted and assayed for enzymaticactivity as described above.

Example 94: In the following hypothetical example and in a mannersimilar to that described above, HA50 is converted to the correspondingcarbohydrate oxazoline with CDMBI, which is subsequently reacted withcholine oxidase for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by the HA50oxazoline occurs. The glycosylated choline oxidase is desalted andassayed for enzymatic activity as described above.

Example 95: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with SOx for about 16 hours at room temperature.SDS-PAGE electrophoresis of the resulting modified protein shows adiscernible shift indicating that glycosylation of the native protein bythe partially deacetylated chitin oxazoline occurs. The glycosylated SOxis desalted and assayed for enzymatic activity as described above.

Example 96: In the following hypothetical example and in a mannersimilar to that described above, HA50 is converted to the correspondingcarbohydrate oxazoline with CDMBI, which is subsequently reacted withMBP-COx2 for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by the HA50oxazoline occurs. The glycosylated MBP-COx2 is desalted and assayed forenzymatic activity as described above.

Example 97: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with MBP-COx2 for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by the partially deacetylated chitin oxazoline occurs. Theglycosylated MBP-COx2 is desalted and assayed for enzymatic activity asdescribed above.

Example 98: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with xanthine oxidase for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by the partially deacetylated chitin oxazoline occurs. Theglycosylated xanthine oxidase is desalted and assayed for enzymaticactivity as described above.

Example 99: In the following hypothetical example and in a mannersimilar to that described above, HA50 is converted to the correspondingcarbohydrate oxazoline with CDMBI, which is subsequently reacted withxanthine oxidase for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by the HA50oxazoline occurs. The glycosylated xanthine oxidase is desalted andassayed for enzymatic activity as described above.

Example 100: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with MBP-xanthine oxidase for about 16 hours atroom temperature. SDS-PAGE electrophoresis of the resulting modifiedprotein shows a discernible shift indicating that glycosylation of thenative protein by the partially deacetylated chitin oxazoline occurs.The glycosylated MBP-xanthine oxidase is desalted and assayed forenzymatic activity as described above.

Example 101: In the following hypothetical example and in a mannersimilar to that described above, HA50 is converted to the correspondingcarbohydrate oxazoline with CDMBI, which is subsequently reacted withxanthine oxidase for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by the HA50oxazoline occurs. The glycosylated MBP-xanthine oxidase is desalted andassayed for enzymatic activity as described above.

Example 102: In the following hypothetical example and in a mannersimilar to that described above, HA50 is converted to the correspondingcarbohydrate oxazoline with CDMBI, which is subsequently reacted withCOx1 for about 16 hours at room temperature. SDS-PAGE electrophoresis ofthe resulting modified protein shows a discernible shift indicating thatglycosylation of the native protein by the HA50 oxazoline occurs. Theglycosylated COx1 is desalted and assayed for enzymatic activity asdescribed above.

Example 103: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with COx1 for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by the partially deacetylated chitin oxazoline occurs. Theglycosylated COx1 is desalted and assayed for enzymatic activity asdescribed above.

Example 104: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with MBP-COx1 for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by the partially deacetylated chitin oxazoline occurs. Theglycosylated MBP-COx1 is desalted and assayed for enzymatic activity asdescribed above.

Example 105: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with COx2 for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by the partially deacetylated chitin oxazoline occurs. Theglycosylated COx2 is desalted and assayed for enzymatic activity asdescribed above.

Example 106: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with ADH for about 16 hours at room temperature.SDS-PAGE electrophoresis of the resulting modified protein shows adiscernible shift indicating that glycosylation of the native protein bythe partially deacetylated chitin oxazoline occurs. The glycosylated ADHis desalted and assayed for enzymatic activity as described above.

Example 107: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with GDH for about 16 hours at room temperature.SDS-PAGE electrophoresis of the resulting modified protein shows adiscernible shift indicating that glycosylation of the native protein bythe partially deacetylated chitin oxazoline occurs. The glycosylated GDHis desalted and assayed for enzymatic activity as described above.

Example 108: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with LDH for about 16 hours at room temperature.SDS-PAGE electrophoresis of the resulting modified protein shows adiscernible shift indicating that glycosylation of the native protein bythe partially deacetylated chitin oxazoline occurs. The glycosylated LDHis desalted and assayed for enzymatic activity as described above.

Example 109: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with CortDH for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by the partially deacetylated chitin oxazoline occurs. Theglycosylated CortDH is desalted and assayed for enzymatic activity asdescribed above.

Example 110: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with MDH for about 16 hours at room temperature.SDS-PAGE electrophoresis of the resulting modified protein shows adiscernible shift indicating that glycosylation of the native protein bythe partially deacetylated chitin oxazoline occurs. The glycosylated MDHis desalted and assayed for enzymatic activity as described above.

Example 111: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MDH for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated MDH is desalted and assayed for enzymaticactivity as described above.

Example 112: In the following hypothetical example and in a mannersimilar to that described above, G2FS2((2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MDH for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,6)occurs. The glycosylated MDH is desalted and assayed for enzymaticactivity as described above.

Example 113: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with LDH for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated LDH is desalted and assayed for enzymaticactivity as described above.

Example 114: In the following hypothetical example and in a mannersimilar to that described above, G2FS2((2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with LDH for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,6)occurs. The glycosylated LDH is desalted and assayed for enzymaticactivity as described above.

Example 115: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with GDH for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated GDH is desalted and assayed for enzymaticactivity as described above.

Example 116: In the following hypothetical example and in a mannersimilar to that described above, G2FS2((2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with GDH for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,6)occurs. The glycosylated GDH is desalted and assayed for enzymaticactivity as described above.

Example 117: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with CortDH for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated CortDH is desalted and assayed for enzymaticactivity as described above.

Example 118: In the following hypothetical example and in a mannersimilar to that described above, G2FS2((2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with CortDH for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2((2,6)occurs. The glycosylated CortDH is desalted and assayed for enzymaticactivity as described above.

Example 119: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with ADH for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated ADH is desalted and assayed for enzymaticactivity as described above.

Example 120: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with ADH for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,6)occurs. The glycosylated ADH is desalted and assayed for enzymaticactivity as described above.

Example 121: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with GOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated GOx is desalted and assayed for enzymaticactivity as described above.

Example 122: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with GOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,6)occurs. The glycosylated GOx is desalted and assayed for enzymaticactivity as described above.

Example 123: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-GOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated MBP-GOx is desalted and assayed for enzymaticactivity as described above.

Example 124: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-GOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,6)occurs. The glycosylated MBP-GOx is desalted and assayed for enzymaticactivity as described above.

Example 125: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with LOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated LOx is desalted and assayed for enzymaticactivity as described above.

Example 126: In the following hypothetical example and in a mannersimilar to that described above, G2FS2((2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with LOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,6)occurs. The glycosylated LOx is desalted and assayed for enzymaticactivity as described above.

Example 127: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-LOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated MBP-LOx is desalted and assayed for enzymaticactivity as described above.

Example 128: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-LOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,6)occurs. The glycosylated MBP-LOx is desalted and assayed for enzymaticactivity as described above.

Example 129: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with COx1 for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated COx1 is desalted and assayed for enzymaticactivity as described above.

Example 130: In the following hypothetical example and in a mannersimilar to that described above, G2FS2((2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with COx1 for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,6)occurs. The glycosylated COx1 is desalted and assayed for enzymaticactivity as described above.

Example 131: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-COx1 for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated MBP-COx1 is desalted and assayed for enzymaticactivity as described above.

Example 132: In the following hypothetical example and in a mannersimilar to that described above, G2FS2((2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-COx1 for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,6)occurs. The glycosylated MBP-COx1 is desalted and assayed for enzymaticactivity as described above.

Example 133: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with COx2 for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated COx2 is desalted and assayed for enzymaticactivity as described above.

Example 134: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with COx2 for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,6)occurs. The glycosylated COx2 is desalted and assayed for enzymaticactivity as described above.

Example 135: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-COx2 for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated MBP-COx2 is desalted and assayed for enzymaticactivity as described above.

Example 136: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-COx2 for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,6)occurs. The glycosylated MBP-COx2 is desalted and assayed for enzymaticactivity as described above.

Example 137: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with AOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated AOx is desalted and assayed for enzymaticactivity as described above.

Example 138: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with AOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,6)occurs. The glycosylated AOx is desalted and assayed for enzymaticactivity as described above.

Example 139: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-AOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated MBP-AOx is desalted and assayed for enzymaticactivity as described above.

Example 140: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-AOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,6)occurs. The glycosylated MBP-AOx is desalted and assayed for enzymaticactivity as described above.

Example 141: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with GlutOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated GlutOx is desalted and assayed for enzymaticactivity as described above.

Example 142: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with GlutOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,6)occurs. The glycosylated GlutOx is desalted and assayed for enzymaticactivity as described above.

Example 143: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-GlutOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated MBP-GlutOx is desalted and assayed forenzymatic activity as described above.

Example 144: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-GlutOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,6)occurs. The glycosylated MBP-GlutOx is desalted and assayed forenzymatic activity as described above.

Example 145: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with SOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated SOx is desalted and assayed for enzymaticactivity as described above.

Example 146: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with SOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,6)occurs. The glycosylated SOx is desalted and assayed for enzymaticactivity as described above.

Example 147: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-SOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated MBP-SOx is desalted and assayed for enzymaticactivity as described above.

Example 148: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-SOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,6)occurs. The glycosylated MBP-SOx is desalted and assayed for enzymaticactivity as described above.

Example 149: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with MBP-SOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by the partially deacetylated chitin oxazoline occurs. Theglycosylated MBP-SOx is desalted and assayed for enzymatic activity asdescribed above.

Example 150: In the following hypothetical example and in a mannersimilar to that described above, HA50 is converted to the correspondingcarbohydrate oxazoline with CDMBI, which is subsequently reacted withMBP-SOx for about 16 hours at room temperature. SDS-PAGE electrophoresisof the resulting modified protein shows a discernible shift indicatingthat glycosylation of the native protein by the HA50 oxazoline occurs.The glycosylated MBP-SOx is desalted and assayed for enzymatic activityas described above.

Example 151: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with NicOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated NicOx is desalted and assayed for enzymaticactivity as described above.

Example 152: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with NicOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,6)occurs. The glycosylated NicOx is desalted and assayed for enzymaticactivity as described above.

Example 153: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-NicOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,3)occurs. The glycosylated MBP-NicOx is desalted and assayed for enzymaticactivity as described above.

Example 154: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-NicOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by G2FS2(α2,6)occurs. The glycosylated MBP-NicOx is desalted and assayed for enzymaticactivity as described above.

Example 155: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with NicOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by the partially deacetylated chitin oxazoline occurs. Theglycosylated NicOx is desalted and assayed for enzymatic activity asdescribed above.

Example 156: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with MBP-NicOx for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by the partially deacetylated chitin oxazoline occurs. Theglycosylated MBP-NicOx is desalted and assayed for enzymatic activity asdescribed above.

Example 157: In the following hypothetical example and in a mannersimilar to that described above, HA50 is converted to the correspondingcarbohydrate oxazoline with CDMBI, which is subsequently reacted withMBP-NicOx for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by the HA50oxazoline occurs. The glycosylated MBP-NicOx is desalted and assayed forenzymatic activity as described above.

Example 158: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with xanthine oxidase for about 16 hours at room temperature.SDS-PAGE electrophoresis of the resulting modified protein shows adiscernible shift indicating that glycosylation of the native protein byG2FS2(α2,3) occurs. The glycosylated xanthine oxidase is desalted andassayed for enzymatic activity as described above.

Example 159: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with xanthine oxidase for about 16 hours at room temperature.SDS-PAGE electrophoresis of the resulting modified protein shows adiscernible shift indicating that glycosylation of the native protein byG2FS2(α2,6) occurs. The glycosylated xanthine oxidase is desalted andassayed for enzymatic activity as described above.

Example 160: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-xanthine oxidase for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by G2FS2(α2,3) occurs. The glycosylated MBP-xanthine oxidase isdesalted and assayed for enzymatic activity as described above.

Example 161: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-xanthine oxidase for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by G2FS2(α2,6) occurs. The glycosylated MBP-xanthine oxidase isdesalted and assayed for enzymatic activity as described above.

Example 162: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with histamine oxidase for about 16 hours atroom temperature. SDS-PAGE electrophoresis of the resulting modifiedprotein shows a discernible shift indicating that glycosylation of thenative protein by the partially deacetylated chitin oxazoline occurs.The glycosylated histamine oxidase is desalted and assayed for enzymaticactivity as described above.

Example 163: In the following hypothetical example and in a mannersimilar to that described above, HA50 is converted to the correspondingcarbohydrate oxazoline with CDMBI, which is subsequently reacted withhistamine oxidase for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by the HA50oxazoline occurs. The glycosylated histamine oxidase is desalted andassayed for enzymatic activity as described above.

Example 164: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with histamine oxidase for about 16 hours at room temperature.SDS-PAGE electrophoresis of the resulting modified protein shows adiscernible shift indicating that glycosylation of the native protein byG2FS2(α2,3) occurs. The glycosylated histamine oxidase is desalted andassayed for enzymatic activity as described above.

Example 165: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with histamine oxidase for about 16 hours at room temperature.SDS-PAGE electrophoresis of the resulting modified protein shows adiscernible shift indicating that glycosylation of the native protein byG2FS2(α2,6) occurs. The glycosylated histamine oxidase is desalted andassayed for enzymatic activity as described above.

Example 166: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with MBP-histamine oxidase for about 16 hours atroom temperature. SDS-PAGE electrophoresis of the resulting modifiedprotein shows a discernible shift indicating that glycosylation of thenative protein by the partially deacetylated chitin oxazoline occurs.The glycosylated MBP-histamine oxidase is desalted and assayed forenzymatic activity as described above.

Example 167: In the following hypothetical example and in a mannersimilar to that described above, HA50 is converted to the correspondingcarbohydrate oxazoline with CDMBI, which is subsequently reacted withMBP-histamine oxidase for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by the HA50oxazoline occurs. The glycosylated MBP-histamine oxidase is desalted andassayed for enzymatic activity as described above.

Example 168: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-histamine oxidase for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by G2FS2(α2,3) occurs. The glycosylated MBP-histamine oxidase isdesalted and assayed for enzymatic activity as described above.

Example 169: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-histamine oxidase for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by G2FS2(α2,6) occurs. The glycosylated MBP-histamine oxidase isdesalted and assayed for enzymatic activity as described above.

Example 170: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with NADH oxidase for about 16 hours at room temperature.SDS-PAGE electrophoresis of the resulting modified protein shows adiscernible shift indicating that glycosylation of the native protein byG2FS2(α2,3) occurs. The glycosylated NADH oxidase is desalted andassayed for enzymatic activity as described above.

Example 171: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with NADH oxidase for about 16 hours at room temperature.SDS-PAGE electrophoresis of the resulting modified protein shows adiscernible shift indicating that glycosylation of the native protein byG2FS2(α2,6) occurs. The glycosylated NADH oxidase is desalted andassayed for enzymatic activity as described above.

Example 172: In the following hypothetical example and in a mannersimilar to that described above, partially deacetylated chitin isconverted to the corresponding carbohydrate oxazoline with CDMBI, whichis subsequently reacted with MBP-NADH oxidase for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by the partially deacetylated chitin oxazoline occurs. Theglycosylated MBP-NADH oxidase is desalted and assayed for enzymaticactivity as described above.

Example 173: In the following hypothetical example and in a mannersimilar to that described above, HA50 is converted to the correspondingcarbohydrate oxazoline with CDMBI, which is subsequently reacted withMBP-histamine oxidase for about 16 hours at room temperature. SDS-PAGEelectrophoresis of the resulting modified protein shows a discernibleshift indicating that glycosylation of the native protein by the HA50oxazoline occurs. The glycosylated MBP-NADH oxidase is desalted andassayed for enzymatic activity as described above.

Example 174: In the following hypothetical example and in a mannersimilar to that described above, G2FS2(α2,3) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-histamine oxidase for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by G2FS2(α2,3) occurs. The glycosylated MBP-NADH oxidase isdesalted and assayed for enzymatic activity as described above.

Example 175: In the following hypothetical example and in a mannersimilar to that described above, G2FS2((2,6) is converted to thecorresponding carbohydrate oxazoline with CDMBI, which is subsequentlyreacted with MBP-histamine oxidase for about 16 hours at roomtemperature. SDS-PAGE electrophoresis of the resulting modified proteinshows a discernible shift indicating that glycosylation of the nativeprotein by G2FS2(α2,6) occurs. The glycosylated MBP-NADH oxidase isdesalted and assayed for enzymatic activity as described above.

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We claim:
 1. An amperometric biosensor comprising: a counter electrode, a reference electrode, one or more optional rejection layers, and a working electrode comprising a sensing element comprising: a support having a surface; and a layer on said surface, said layer comprising a glycosylated protein, said glycosylated protein comprising a starting protein having an amino sidechain with a nucleophilic moiety, said amino side chain selected from the group consisting of lysine, histidine, and arginine, said nucleophilic moiety being N, said starting protein glycosylated with a carbohydrate having an oxazoline moiety on the reducing end thereof, said oxazoline moiety covalently bound with said nucleophilic N moiety, and said glycosylated protein having a molecular weight of at least 7,500 Daltons.
 2. The glycosylated protein of claim 1, wherein said glycosylated protein is an enzyme.
 3. The glycosylated protein of claim 2, wherein said enzyme is selected from the group consisting of oxidase, oxidoreductase, and dehydrogenase enzymes.
 4. The biosensor of claim 2, wherein said enzyme is independently selected from the group consisting of glucose oxidases, lactate oxidases, alcohol oxidases, glutamate oxidases, xanthine oxidases, sarcosine oxidases, cholesterol oxidases, oxalate oxidases, D-amino acid oxidases, choline oxidases, glutathione sulfhydryl oxidases, (S)-6-hydroxynicotine oxidases, (R)-6-hydroxynicotine oxidases, nicotine oxidases, pyruvate oxidases, acyl coenzyme A oxidases, glycerolphosphate oxidases, GABA oxidases, histamine oxidases, diamine oxidases, nucleoside oxidases, L-lysine oxidases, L-aspartate oxidases, glycine oxidases, galactose oxidases, NADH oxidases, urate oxidases, histamine oxidases, succinate oxidases, glucose dehydrogenases, alcohol dehydrogenases, cortisol dehydrogenases, lactate dehydrogenases, histamine dehydrogenases, xanthine dehydrogenases, succinate dehydrogenases, nicotine dehydrogenases, sarcosine dehydrogenases, 6-hydroxypseudooxynicotine dehydrogenase, and fusion proteins thereof.
 5. The glycosylated protein of claim 1, wherein said carbohydrate has a molecular weight of at least 200 Daltons.
 6. The glycosylated protein of claim 5, wherein said carbohydrate has a molecular weight of at least 10,000 Daltons.
 7. The glycosylated protein of claim 6, wherein said carbohydrate has a molecular weight of at least 25,000 Daltons.
 8. The glycosylated protein of claim 1, wherein said carbohydrate is linear or branched.
 9. The glycosylated protein of claim 1, wherein said carbohydrate is selected from the group consisting of chitin, partially deacylated chitin, hyaluronic acid, keratin, keratin sulfate, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, heparin, native N-glycan cores, high-mannose N-glycans, hybrid N-glycans, complex N-glycans, and derivatives thereof.
 10. The glycosylated protein of claim 1, said glycosylated protein having one or both of the tautomeric forms

wherein each of R₁, R₂, and R₃ is individually and independently selected from the group consisting of H and saccharides, and wherein R is selected from the group consisting of C1-C6 alkyl, branched C3-C8 alkyl, —(CH₂)_(m)—CN, —(CH₂)_(m)OR6, —(CH₂)_(m)—CO₂H, —(CH₂)_(m)—CO₂R6, —(CH₂)_(m)—NR6(R7), —(CH₂)_(m)—S(O)_(n)—C1-C6 alkyl, —(CH₂)_(m)—C(O)NR6(R7), —(CH₂)_(m)—CO₂—C4-C6 heterocyclyl, —(CH₂)_(m)—C4-C6 heterocyclyl, —(CH₂)_(m)—CO₂—C4-C6 heteroaryl, or —(CH₂)_(m)—C4-C6-heteroaryl, wherein each alkyl may optionally contain an ether linkage and, wherein each alkyl is optionally substituted with one or two C1-C6 alkyl groups, each R6 and R7 is individually and independently H, C1-C6 alkyl, or branched C3-C8 alkyl, each m is individually and independently 1, 2, 3, 4, or 5, each n is individually and independently 0, 1, or
 2. 11. The biosensor of claim 1, wherein said glycosylated protein is selected from the group consisting of glucose oxidases modified with hyaluronic acid, glucose oxidases modified with chitin, glucose oxidases modified with partially deacetylated chitin, glucose oxidases modified with high-mannose N-glycans, glucose oxidases modified with keratin, glucose oxidases modified with keratin sulfate, glucose oxidases modified with chondroitin, glucose oxidases modified with chondroitin sulfate, glucose oxidases modified with dermatan, glucose oxidases modified with dermatan sulfate, glucose oxidases modified with heparin, glucose oxidases modified with hybrid N-glycans, glucose oxidases modified with complex N-glycans, lactate oxidases modified with hyaluronic acid, lactate oxidases modified with chitin, lactate oxidases modified with partially deacetylated chitin, lactate oxidases modified with high-mannose N-glycans, lactate oxidases modified with keratin, lactate oxidases modified with keratin sulfate, lactate oxidases modified with chondroitin, lactate oxidases modified with chondroitin sulfate, lactate oxidases modified with dermatan, lactate oxidases modified with dermatan sulfate, lactate oxidases modified with heparin, lactate oxidases modified with hybrid N-glycans, lactate oxidases modified with complex N-glycans, alcohol oxidases modified with hyaluronic acid, alcohol oxidases modified with chitin, alcohol oxidases modified with partially deacetylated chitin, alcohol oxidases modified with high-mannose N-glycans, alcohol oxidases modified with keratin, alcohol oxidases modified with keratin sulfate, alcohol oxidases modified with chondroitin, alcohol oxidases modified with chondroitin sulfate, alcohol oxidases modified with dermatan, alcohol oxidases modified with dermatan sulfate, alcohol oxidases modified with heparin, alcohol oxidases modified with hybrid N-glycans, alcohol oxidases modified with complex N-glycans, glutamate oxidases modified with hyaluronic acid, glutamate oxidases modified with chitin, glutamate oxidases modified with partially deacetylated chitin, glutamate oxidases modified with high-mannose N-glycans, glutamate oxidases modified with keratin, glutamate oxidases modified with keratin sulfate, glutamate oxidases modified with chondroitin, glutamate oxidases modified with chondroitin sulfate, glutamate oxidases modified with dermatan, glutamate oxidases modified with dermatan sulfate, glutamate oxidases modified with heparin, glutamate oxidases modified with hybrid N-glycans, glutamate oxidases modified with complex N-glycans, choline oxidases modified with hyaluronic acid, choline oxidases modified with chitin, choline oxidases modified with partially deacetylated chitin, choline oxidases modified with high-mannose N-glycans, choline oxidases modified with keratin, choline oxidases modified with keratin sulfate, choline oxidases modified with chondroitin, choline oxidases modified with chondroitin sulfate, choline oxidases modified with dermatan, choline oxidases modified with dermatan sulfate, choline oxidases modified with heparin, choline oxidases modified with hybrid N-glycans, choline oxidases modified with complex N-glycans, sarcosine oxidases modified with hyaluronic acid, sarcosine oxidases modified with chitin, sarcosine oxidases modified with partially deacetylated chitin, sarcosine oxidases modified with high-mannose N-glycans, sarcosine oxidases modified with keratin, sarcosine oxidases modified with keratin sulfate, sarcosine oxidases modified with chondroitin, sarcosine oxidases modified with chondroitin sulfate, sarcosine oxidases modified with dermatan, sarcosine oxidases modified with dermatan sulfate, sarcosine oxidases modified with heparin, sarcosine oxidases modified with hybrid N-glycans, sarcosine oxidases modified with complex N-glycans, xanthine oxidases modified with hyaluronic acid, xanthine oxidases modified with chitin, xanthine oxidases modified with partially deacetylated chitin, xanthine oxidases modified with high-mannose N-glycans, xanthine oxidases modified with keratin, xanthine oxidases modified with keratin sulfate, xanthine oxidases modified with chondroitin, xanthine oxidases modified with chondroitin sulfate, xanthine oxidases modified with dermatan, xanthine oxidases modified with dermatan sulfate, xanthine oxidases modified with heparin, xanthine oxidases modified with hybrid N-glycans, xanthine oxidases modified with complex N-glycans, oxalate oxidases modified with hyaluronic acid, oxalate oxidases modified with chitin, oxalate oxidases modified with partially deacetylated chitin, oxalate oxidases modified with high-mannose N-glycans, oxalate oxidases modified with keratin, oxalate oxidases modified with keratin sulfate, oxalate oxidases modified with chondroitin, oxalate oxidases modified with chondroitin sulfate, oxalate oxidases modified with dermatan, oxalate oxidases modified with dermatan sulfate, oxalate oxidases modified with heparin, oxalate oxidases modified with hybrid N-glycans, oxalate oxidases modified with complex N-glycans, cholesterol oxidases modified with hyaluronic acid, cholesterol oxidases modified with chitin, cholesterol oxidases modified with partially deacetylated chitin, cholesterol oxidases modified with high-mannose N-glycans, cholesterol oxidases modified with keratin, cholesterol oxidases modified with keratin sulfate, cholesterol oxidases modified with chondroitin, cholesterol oxidases modified with chondroitin sulfate, cholesterol oxidases modified with dermatan, cholesterol oxidases modified with dermatan sulfate, cholesterol oxidases modified with heparin, cholesterol oxidases modified with hybrid N-glycans, cholesterol oxidases modified with complex N-glycans, histamine oxidases modified with hyaluronic acid, histamine oxidases modified with chitin, histamine oxidases modified with partially deacetylated chitin, histamine oxidases modified with high-mannose N-glycans, histamine oxidases modified with keratin, histamine oxidases modified with keratin sulfate, histamine oxidases modified with chondroitin, histamine oxidases modified with chondroitin sulfate, histamine oxidases modified with dermatan, histamine oxidases modified with dermatan sulfate, histamine oxidases modified with heparin, histamine oxidases modified with hybrid N-glycans, histamine oxidases modified with complex N-glycans, glycine oxidases modified with hyaluronic acid, glycine oxidases modified with chitin, glycine oxidases modified with partially deacetylated chitin, glycine oxidases modified with high-mannose N-glycans, glycine oxidases modified with keratin, glycine oxidases modified with keratin sulfate, glycine oxidases modified with chondroitin, glycine oxidases modified with chondroitin sulfate, glycine oxidases modified with dermatan, glycine oxidases modified with dermatan sulfate, glycine oxidases modified with heparin, glycine oxidases modified with hybrid N-glycans, glycine oxidases modified with complex N-glycans, NADH oxidases modified with hyaluronic acid, NADH oxidases modified with chitin, NADH oxidases modified with partially deacetylated chitin, NADH oxidases modified with high-mannose N-glycans, NADH oxidases modified with keratin, NADH oxidases modified with keratin sulfate, NADH oxidases modified with chondroitin, NADH oxidases modified with chondroitin sulfate, NADH oxidases modified with dermatan, NADH oxidases modified with dermatan sulfate, NADH oxidases modified with heparin, NADH oxidases modified with hybrid N-glycans, NADH oxidases modified with complex N-glycans, galactose oxidases modified with hyaluronic acid, galactose oxidases modified with chitin, galactose oxidases modified with partially deacetylated chitin, galactose oxidases modified with high-mannose N-glycans, galactose oxidases modified with keratin, galactose oxidases modified with keratin sulfate, galactose oxidases modified with chondroitin, galactose oxidases modified with chondroitin sulfate, galactose oxidases modified with dermatan, galactose oxidases modified with dermatan sulfate, galactose oxidases modified with heparin, galactose oxidases modified with hybrid N-glycans, galactose oxidases modified with complex N-glycans, alcohol dehydrogenases modified with hyaluronic acid, alcohol dehydrogenases modified with chitin, alcohol dehydrogenases modified with partially deacetylated chitin, alcohol dehydrogenases modified with high-mannose N-glycans, alcohol dehydrogenases modified with keratin, alcohol dehydrogenases modified with keratin sulfate, alcohol dehydrogenases modified with chondroitin, alcohol dehydrogenases modified with chondroitin sulfate, alcohol dehydrogenases modified with dermatan, alcohol dehydrogenases modified with dermatan sulfate, alcohol dehydrogenases modified with heparin, alcohol dehydrogenases modified with hybrid N-glycans, alcohol dehydrogenases modified with complex N-glycans, glucose dehydrogenases modified with hyaluronic acid, glucose dehydrogenases modified with chitin, glucose dehydrogenases modified with partially deacetylated chitin, glucose dehydrogenases modified with high-mannose N-glycans, glucose dehydrogenases modified with keratin, glucose dehydrogenases modified with keratin sulfate, glucose dehydrogenases modified with chondroitin, glucose dehydrogenases modified with chondroitin sulfate, glucose dehydrogenases modified with dermatan, glucose dehydrogenases modified with dermatan sulfate, glucose dehydrogenases modified with heparin, glucose dehydrogenases modified with hybrid N-glycans, glucose dehydrogenases modified with complex N-glycans, L-lactate dehydrogenases modified with hyaluronic acid, L-lactate dehydrogenases modified with chitin, L-lactate dehydrogenases modified with partially deacetylated chitin, L-lactate dehydrogenases modified with high-mannose N-glycans, L-lactate dehydrogenases modified with keratin, L-lactate dehydrogenases modified with keratin sulfate, L-lactate dehydrogenases modified with chondroitin, L-lactate dehydrogenases modified with chondroitin sulfate, L-lactate dehydrogenases modified with dermatan, L-lactate dehydrogenases modified with dermatan sulfate, L-lactate dehydrogenases modified with heparin, L-lactate dehydrogenases modified with hybrid N-glycans, L-lactate dehydrogenases modified with complex N-glycans, cortisol dehydrogenases modified with hyaluronic acid, cortisol dehydrogenases modified with chitin, cortisol dehydrogenases modified with partially deacetylated chitin, cortisol dehydrogenases modified with high-mannose N-glycans, cortisol dehydrogenases modified with keratin, cortisol dehydrogenases modified with keratin sulfate, cortisol dehydrogenases modified with chondroitin, cortisol dehydrogenases modified with chondroitin sulfate, cortisol dehydrogenases modified with dermatan, cortisol dehydrogenases modified with dermatan sulfate, cortisol dehydrogenases modified with heparin, cortisol dehydrogenases modified with hybrid N-glycans, cortisol dehydrogenases modified with complex N-glycans, galactose dehydrogenases modified with hyaluronic acid, galactose dehydrogenases modified with chitin, galactose dehydrogenases modified with partially deacetylated chitin, galactose dehydrogenases modified with high-mannose N-glycans, galactose dehydrogenases modified with keratin, galactose dehydrogenases modified with keratin sulfate, galactose dehydrogenases modified with chondroitin, galactose dehydrogenases modified with chondroitin sulfate, galactose dehydrogenases modified with dermatan, galactose dehydrogenases modified with dermatan sulfate, galactose dehydrogenases modified with heparin, galactose dehydrogenases modified with hybrid N-glycans, galactose dehydrogenases modified with complex N-glycans, glycerol dehydrogenases modified with hyaluronic acid, glycerol dehydrogenases modified with chitin, glycerol dehydrogenases modified with partially deacetylated chitin, glycerol dehydrogenases modified with high-mannose N-glycans, glycerol dehydrogenases modified with keratin, glycerol dehydrogenases modified with keratin sulfate, glycerol dehydrogenases modified with chondroitin, glycerol dehydrogenases modified with chondroitin sulfate, glycerol dehydrogenases modified with dermatan, glycerol dehydrogenases modified with dermatan sulfate, glycerol dehydrogenases modified with heparin, glycerol dehydrogenases modified with hybrid N-glycans, glycerol dehydrogenases modified with complex N-glycans, glucose-6-phosphate dehydrogenases modified with hyaluronic acid, glucose-6-phosphate dehydrogenases modified with chitin, glucose-6-phosphate dehydrogenases modified with partially deacetylated chitin, glucose-6-phosphate dehydrogenases modified with high-mannose N-glycans, glucose-6-phosphate dehydrogenases modified with keratin, glucose-6-phosphate dehydrogenases modified with keratin sulfate, glucose-6-phosphate dehydrogenases modified with chondroitin, glucose-6-phosphate dehydrogenases modified with chondroitin sulfate, glucose-6-phosphate dehydrogenases modified with dermatan, glucose-6-phosphate dehydrogenases modified with dermatan sulfate, glucose-6-phosphate dehydrogenases modified with heparin, glucose-6-phosphate dehydrogenases modified with hybrid N-glycans, glucose-6-phosphate dehydrogenases modified with complex N-glycans, 3-hydroxybutyrate dehydrogenases modified with hyaluronic acid, 3-hydroxybutyrate dehydrogenases modified with chitin, 3-hydroxybutyrate dehydrogenases modified with partially deacetylated chitin, 3-hydroxybutyrate dehydrogenases modified with high-mannose N-glycans, 3-hydroxybutyrate dehydrogenases modified with keratin, 3-hydroxybutyrate dehydrogenases modified with keratin sulfate, 3-hydroxybutyrate dehydrogenases modified with chondroitin, 3-hydroxybutyrate dehydrogenases modified with chondroitin sulfate, 3-hydroxybutyrate dehydrogenases modified with dermatan, 3-hydroxybutyrate dehydrogenases modified with dermatan sulfate, 3-hydroxybutyrate dehydrogenases modified with heparin, 3-hydroxybutyrate dehydrogenases modified with hybrid N-glycans, 3-hydroxybutyrate dehydrogenases modified with complex N-glycans, L-malate dehydrogenases modified with hyaluronic acid, L-malate dehydrogenases modified with chitin, L-malate dehydrogenases modified with partially deacetylated chitin, L-malate dehydrogenases modified with high-mannose N-glycans, L-malate dehydrogenases modified with keratin, L-malate dehydrogenases modified with keratin sulfate, L-malate dehydrogenases modified with chondroitin, L-malate dehydrogenases modified with chondroitin sulfate, L-malate dehydrogenases modified with dermatan, L-malate dehydrogenases modified with dermatan sulfate, L-malate dehydrogenases modified with heparin, L-malate dehydrogenases modified with hybrid N-glycans, L-malate dehydrogenases modified with complex N-glycans, sorbitol dehydrogenases modified with hyaluronic acid, sorbitol dehydrogenases modified with chitin, sorbitol dehydrogenases modified with partially deacetylated chitin, sorbitol dehydrogenases modified with high-mannose N-glycans, sorbitol dehydrogenases modified with keratin, sorbitol dehydrogenases modified with keratin sulfate, sorbitol dehydrogenases modified with chondroitin, sorbitol dehydrogenases modified with chondroitin sulfate, sorbitol dehydrogenases modified with dermatan, sorbitol dehydrogenases modified with dermatan sulfate, sorbitol dehydrogenases modified with heparin, sorbitol dehydrogenases modified with hybrid N-glycans, and sorbitol dehydrogenases modified with complex N-glycans, and fusion proteins thereof.
 12. The biosensor of claim 11, wherein said glycosylated protein is selected from the group consisting of glucose oxidases modified with hyaluronic acid, glucose oxidases modified with partially deacetylated chitin, glucose oxidases modified with complex N-glycans, lactate oxidases modified with hyaluronic acid, lactate oxidases modified with partially deacetylated chitin, lactate oxidases modified with complex N-glycans, alcohol oxidases modified with hyaluronic acid, alcohol oxidases modified with partially deacetylated chitin, alcohol oxidases modified with complex N-glycans, glutamate oxidases modified with hyaluronic acid, glutamate oxidases modified with partially deacetylated chitin, glutamate oxidases modified with complex N-glycans, choline oxidases modified with hyaluronic acid, choline oxidases modified with partially deacetylated chitin, choline oxidases modified with complex N-glycans, sarcosine oxidases modified with hyaluronic acid, sarcosine oxidases modified with partially deacetylated chitin, sarcosine oxidases modified with complex N-glycans, xanthine oxidases modified with hyaluronic acid, xanthine oxidases modified with partially deacetylated chitin, xanthine oxidases modified with complex N-glycans, cholesterol oxidases modified with hyaluronic acid, cholesterol oxidases modified with partially deacetylated chitin, cholesterol oxidases modified with complex N-glycans, histamine oxidases modified with hyaluronic acid, histamine oxidases modified with partially deacetylated chitin, histamine oxidases modified with complex N-glycans, NADH oxidases modified with hyaluronic acid, NADH oxidases modified with partially deacetylated chitin, NADH oxidases modified with complex N-glycans, alcohol dehydrogenases modified with hyaluronic acid, alcohol dehydrogenases modified with partially deacetylated chitin, alcohol dehydrogenases modified with complex N-glycans, glucose dehydrogenases modified with hyaluronic acid, glucose dehydrogenases modified with partially deacetylated chitin, glucose dehydrogenases modified with complex N-glycans, L-lactate dehydrogenases modified with hyaluronic acid, L-lactate dehydrogenases modified with partially deacetylated chitin, L-lactate dehydrogenases modified with complex N-glycans, cortisol dehydrogenases modified with hyaluronic acid, cortisol dehydrogenases modified with partially deacetylated chitin, cortisol dehydrogenases modified with complex N-glycans, L-malate dehydrogenases modified with hyaluronic acid, L-malate dehydrogenases modified with partially deacetylated chitin, and L-malate dehydrogenases modified with complex N-glycans, and fusion proteins thereof.
 13. The biosensor of claim 1, wherein said glycosylated protein is selected from the group consisting of glucose oxidase and glucose dehydrogenase.
 14. The biosensor of claim 1, wherein said glycosylated protein is selected from the group consisting of lactate oxidase and lactate dehydrogenase.
 15. The biosensor of claim 1, wherein said glycosylated protein is selected from the group consisting of nicotine oxidase, nicotine dehydrogenase, (S)-6-hydroxynicotine oxidases, (R)-6-hydroxynicotine oxidases, and 6-hydroxypseudooxynicotine dehydrogenase.
 16. The biosensor of claim 1, wherein said glycosylated protein is selected from the group consisting of histamine oxidase and histamine dehydrogenase.
 17. The biosensor of claim 1, wherein said glycosylated protein is selected from the group consisting of succinate oxidase, succinate dehydrogenase, xanthine oxidase, xanthine dehydrogenase, sarcosine oxidase, and sarcosine dehydrogenase.
 18. The biosensor of claim 1, wherein said glycosylated protein is a cortisol dehydrogenase.
 19. The biosensor of claim 1, wherein said glycosylated protein is a NADH oxidase.
 20. The biosensor of claim 1, wherein said glycosylated protein is an urate oxidase. 